MIOROSCOPIO  BOTANY. 


A  MANUAL  OF  THE  MICROSCOPE 


VEGETABLE  HISTOLOGY. 


BY 

DE.    EDUARD    STRASBURGER. 


FROM    THE    GERMAN 
BY 

REV.    A.    B.    HERVEY 


Ac.  f\fo, 


BOSTON  : 
SAMUEL    E.    CASSINO, 

1887. 


COPYRIGHT,   1.S87, 

1!Y 

SAMUEL  E.  CASSINO, 


TRANSLATOR'S  PREFACE. 


The  "Kleine  Botanische  Practicum"  of  which  this  is  a 
translation  is  an  abridgment  of  a  larger  work  of  the  same 
kind  made  by  the  author,  Dr.  Strasbnrger.  I  have  made 
further  condensation  of  the  work  in  those  chapters  where 
the  matter  and  expression  would  admit,  notal)ly  in  Chapter 
VIII  and  those  immediately  preceding,  and  in  Chapters 
XXX  and  XXXI,  but  in  no  case  have  I  omitted  essential 
matters  from  the  text.  The  Introduction  alone  is  shortened 
by  omission,  because  portions  of  it  were  quite  irrelevant  to 
the  purposes  of  an  American  edition.  For  a  similar  rea- 
son, and  to  save  space,  the  Author's  preface  is  not  repro- 
duced. 

The  first  two  "Registers"  or  Indices,  those  enumerat- 
ing the  plants  and  reagents  used  in  the  studies,  are  also 
omitted,  as  being  neither  essential  nor  very  important, 
the  references  being  all  contained  in  the  general  subject- 
index.  The  formuhe  contained  in  register  II  are  found 
in  an  appendix. 

The  work  is  divided  into  thirty-two  Lessons  or  Chap- 
ters to  adapt  it  to  the  weeks  in  a  German  College  3'ear. 
It  is  believed  that  the  work  is  well  adapted  to  the  needs 
of  both  the  solitar}^  wcn-ker  and    students  in    American 

Colleges. 

A.  B.  Hervey. 

Taunton,  Ma.ss.,  4  July,  1887. 

(iii) 


TABLE  OF  CONTENTS. 


Page. 
Introduction.  1 

Lesson. 

I  Use  of  the  Microscope.      Structure  of  Starch.       6 
II  Gluten.     Fatty  Oils.    Making  Permanent  Prep- 
arations.    Use  of  the  Simple  Microscope.  18 

III  Protoplasm  Streaming.     The  Nucleus.     Draw- 

ing with  the  Camera.     Determining  Magnifi- 
cation. 29 

IV  Chromatophores.     Colored  Cell-sap.  38 
V  Tissue,     Thickening  of  the  Wall.     Reaction  on 

Sugar.     Inulin  Nitrates.   Tannin.  Wood  sub- 
stance or  Lignin.  45 
VI  Epidermis.     Stoniata.                                                   60 
VII  Epidermis.     Hairs.     Wax  and  Mucilage.                71 
VIII  Closed  Collateral  Vascular  Bundles.                          82 
IX  Open  Collateral  Vascular  Bundles.                           95 
X  Structure  of  the  Coniferous  Stems.                          108 
XI  Structure  of  Linden.    Bicollateral  Vascular  Bun- 
dles of  the  Cucurbita.     Sieve  Tubes.                  118 
XII  Vascular  Bundles  of  the  Axile    Cylinder,  and 

the  Secondary  Lateral  Growth  of  the    Roots.  129 

XIII  Vascular  Bundles  of  the  Ferns  and  L3'copods.      138 

XIV  Cork.     Lenticels.  145 
XV  Structure  of  the   Foliage   and   Floral   Leaves. 

The  ends  of  the  Vascular  Bundles.  151 

(V) 


VI  CONTENTS. 

XVI  Vegetative  Cone  of  the  Stem.  161 

XVII  Vegetative  Cone  of  the  Root.  173 

XVIII  Histology  of  the  Mosses.  181 
XIX  Histology  of    the  Fungi,   Lichens,  and   Algae. 

Staining  the  Cell  Contents.  191 

XX  Diatoms.     Protococcus.     Yeast.    Protophytes.  202 

XXI  Schizomycetes.    Use  of  the  Immersion  System.  214 

XXII  Repro<Juction  of  the  Algae.  236 

XXIII  Eeproduction  of  the  Fungi.  246 

XXIV  Reproduction  of  the  Fungi  and  Lichens.  253 
XXV. Reproduction  of  the  Mosses.  263 

XXVI  Reproduction  of  the  Vascular  Crj^ptogaras.  278 

XXVII  Reproduction  of  the  Gymnosperms.  289 

XXVIII  The  Androecium  of  the  Angiosperms.  304 

XXIX  The  Gyneceum  of  the  Angiosperms.  317 

XXX  Structure  of  the  Seeds  of  Angiosperms.  S32 

XXXI  Fruit  of  the  Angiosperms.  341 

XXXII  Self-division  of  Nucleus  and  Cell.  350 


INTliüDUCTION.* 


In  case  the  beginner  should  wish  to  provide  himself 
•with  a  water-immersion  lens,  he  is  recommended  to  get 
one  without  the  screw-collar  adjustment.  He  will  find  it 
more  difficult  to  learn  the  management  of  the  other  sort. 
The  most  skilful  observers  make  no  use  of  this  adjust- 
ment with  the  lower  powers,  and  these  are  the  ones  re- 
ferred to  here. 

Immersion  lenses  without  the  screws-collar  adjustment 
are  corrected  for  a  ü'iven  thickness  of  cover-o-jass  and 
should  be  used  with  that  thickness  only.  Bj  providing 
himself  with  that  glass  he  can  dispense  with  the  correc- 
tion adjustment  even  with  his  high  powers,  and  would 
need  it  only  in  stvidying  objects  already  mounted  under 
cover-glasses  of  other  thicknesses. 

He  who  is  not  afraid  of  a  larger  expenditure  wil]  do 
well  to  buy  a  "homogeneous"  instead  of  a  water-immersion 
system.  These  systems  are  all  without  colhir  correction 
since  the  thickness  of  cover-glass  within  permissible  lim- 
its is  a  matter  of  indifference.  Since  they  will  bear  much 
higher  eye-pieces  than  either  the  dry  or  the  water-immer- 

*  A  considerable  part  of  the  Author's  Introduction  is  given  up  to  the  mere  enu- 
meration of  stands  and  lenses  of  German  and  other  Continental,  and  English  mak- 
ers, compiled  from  their  latest  catalogues.  As  American  students  would  mostly 
buy  Amei'ican  instruments,  or  could  easily  get  the  same  information  from  the  cat- 
alogues used  by  the  author,  it  was  not  tliought  necessary  to  print  tliis  part  of  the 
text.  The  main  import  of  his  teaching  on  this  point  is  to  choose  the  simplest  and 
handiest  stand,  with  lenses  of  medium  power;  such  as  in  this  country  are  gener- 
ally known  as  "Students'  Microscoijes."  The  stands  should  however  be  chosen 
witli  reference  to  the  use  of  the  higher  and  highest  objectives  on  them.  Speaking 
of  water-immersion  lenses  he  continues  as  above.— A.  B.  H. 

1  (1) 


2  INTRODUCTION. 

sion  systems,  as  many  different  magnifications  ma}'^  be  got 
with  one  of  these  as  with  several  of  the  latter.  Homo- 
geneons  immersion  lenses  give  the  best  results  when  used 
with  the  Abbe  illuminating  apparatus.  This  apparatus  is 
applied  only  to  the  larger  and  more  costly  stands.  *  *  * 
Still  the  homogeneous  systems  may  be  used  with  great 
advantage  on  the  smaller  stands. 

It  does  not  come  within  my  purpose  to  propound  a  the- 
ory of  the  production  of  the  microscopic  image,  but  refer 
my  readers  to  text-books  of  physics  and  special  works  on 
the  microscope  for  that  (1).  It  is  my  aim  to  make  the 
beginner  familiar  with  the  more  important  facts  of  micro- 
scopical botany,  with  the  use  of  the  microscope  and  with 
microscopical  technique.  This  can  be  accomplished  only 
by  study.  But  for  convenience  in  comparing  and  consult- 
ing the  various  statements  scattered  throuHi  the  text  a 
detailed  index  is  appended. 

Besides  the  compound  microscope,  a  simple,  or  a  so- 
called  "preparing  microscope"  or  "simplex,"  is  also  neces- 
sary. For  all  the  purposes  contemplated  in  this  l)Ook,  the 
small  preparing  stand  (No.  Ill  of  Zeiss'  Catalogue,  for 
$4.50)  with  a  magnifying  glass  having  a  power  of  from  five 
to  ten  diameters  (No.  112)  for  $1.50,  a  doublet  of  fifteen 
and  another  of  thirty  diameters  of  magnification  (No.  113) 
for  $1.50  each,  are  sufficient.  The  magnifying  glass  em- 
ployed in  this  combination  may  also  be  used  as  a  hand 
magnifier. 

The  compound  microscope  may  be  used  for  a  "preparing 
microscope"  by  uniting  a  prism  with  the  ocular  as.  in  Na- 
cliet's  plan,  or  by  an  "erecting"  ocular  like  that  of  Hart- 
nack.  For  the  use  of  this  contrivance  a  draw-tube  is 
necessary,  the  erecting  prism  being  screwed  into  the  lower 
end  of  it.  The  image  loses  something  of  its  sharpness  but 
still  sufficiently  answers  its  purpose.     A  certain  advantage 


INTRODUCTION.  ö 

is  gained  in  manipulating  very  small  objects  under  the  com- 
pound microscope,  for  they  are  not  lost  from  the  field  of 
view,  and  do  not  have  to  be  transferred  from  the  com- 
pound to  the  simple  microscope  and  back  again  to  find 
them.  Working  with  the  erecting  ocular  oflers  scarcely 
greater  difiiculties  than  with  a  "simplex,"  but  the  erecting 
prism  obliges  one  to  look  obliquely  forward  rather  than 
downward  toward  the  hands,  which  is  somewhat  inconven- 
ient. The  erecting  prism  attached  to  the  ocular  diminishes 
the  field  of  vision  in  most  cases.  Very  low  powers  must 
be  used  for  this  work. 

Another  indispensable  requisite  is  a  good  magnifying 
glass  by  which  to  make  our  preliminary  survey  of  the  ob- 
ject to  be  investigated.  As  before  mentioned,  if  the  pre- 
paring microscope  is  furnished  with  a  good  magnifier  it. 
may  be  used  as  a  hand-lens.  One  with  a  power  of  about 
six  diameters  is  usually  preferred.  The  aplanatic  magni- 
fiers are  very  excellent  and  correspondingly  expensive. 

Fora  drawing  prism  to  be  used  on  the  microscope,  either 
the  new  Abbe  camera  lucida  (Zeiss  Cat.,  No.  64),  or  the 
camera  lucida  with  two  prisms  (Zeiss  Cat,,  No.  65),  is  to 
be  preferred  before  all  others.  The  former  is  fitted  and 
adjusted  to  the  No.  2  ocular.  During  observation  it  is  re- 
moved. It  allows  one  to  draw  on  an  horizontal  surface. 
The  latter  is  ftistened  to  the  tube  or  ocular  with  a  ring. 
The  drawing  is  done  on  an  inclined  surface.  It  may  be 
kept  constantly  on  the  microscope,  being  turned  to  one 
side  during  ol)servation.  The  drawing  table,  either  hori- 
zontal or  inclined  at  an  angle  of  25^  as  the  case  may  be, 
should  be  adjusted  at  the  height  of  the  microscope  stage, 
or  at  the  distance  of  clearest  vision  for  an  especially  near- 
or  far-sighted  observer.* 

One  needs  also  an  objective  micrometer. 

*It  slioiilil  always  be  fixed  ut  a  standard  distauce  of  ten  inches  from  the  focus 
of  the  ocular  .—A.  B.  H. 


4  INTRODUCTION. 

Any  steady  work-table  may  be  used  in  microscopical 
work.  It  shoukl  be  neither  too  small,  nor  have  a  bright 
surface.  Choose  a  window  with  a  free  outlook,  if  possible 
(it  matters  not  in  what  direction) ,  and  place  the  microscope 
IJ  to  2m.  from  it.  If  there  is  direct  smilight,  interpose 
a  white  curtain.  This  glaring  white  light  is  the  best  pos- 
sible for  working  with  high  powers. 

Object-slides  and  cover-glasses  may  be  obtained  of  the 
dealers.  As  between  the  Giessen  form  of  object-slide, 
Avhich  is  48  mm.  h)ng  by  28  mm.  wide,  and  the  English 
which  is  76  by  26  mm.,  the  latter  is  in  man}"  respects  hand- 
ier.     Of  the  cover-glasses,  those  for  common  use  may 


Fig.  1.    Zinc  rack  for  slides.    Usert  under  glass  bell. 

conveniently  be  squares  about  18  mm.  on  a  side,  but  for 
special  purposes  both  smaller  and  larger  forms  may  be 
provided.  If  one  uses  high  powers,  he  should  have  his 
cover-glasses  prepared  of  a  definite  thickness.* 

One  will  also  need  razors  both  flat  and  concave  ;  small 
and  large  steel  forceps  ;  fine-pointed  dissecting  scissors, 
for  which  fine  embroidery  scissors  will  answer ;  a  pair  of 
needle  holders  somewhat  like  a  crochet-needle  holder,  but 
one  in  which  the  finest  sewing  needle  mav  be  held  fast ; 
English  sewing  needles  from  No.  8  upwards  ;  scalpels  ;fine 

*  For  ordinary  use,  circles  are  to  be  preferred  to  squares.  Tliose  IS  mm.  in  di- 
ameter are  perliaps  best  for  examination  of  objects  in  water.  But  for  permanent 
preparation  of  small  oljjects,  circles  of  uniform  size,  15  mm.  in  diameter  should 
be  chosen.— A.  B.  H. 


INTRODUCTION.  5 

camel's  hair  pencils  ;  a  small,  watchmaker's  hand-vice  ;  glass 
tubes  and  rods  ;  watch  glasses  of  ditferent  sizes,  and  glass 
disks  of  corresponding  sizes  with  which  to  cover  them  ; 
low  glass  hells  with  which  to  construct  moist-chambers  ; 
zinc  racks  as  illustrated,  about  one-half  natural  size  in 
Fig.  1,  on  which  to  lay  the  slides  under  the  bells  ;  two  high 
glass  bells  with  which  to  cover  the  simple  and  compound 
microscopes  ;  and,  finally,  elder  pith. 

The  list  of  necessary  reagents  is  appended  at  the  end 
of  this  book. 

For  the  preservation  of  permanent  preparations,  the 
dealers  in  such  goods  furnish  excellent  cabinets,  in  which 
the  objects  lie  flat,  and  can  easily  be  found  and  inspected. 

NOTK. 

(1)  Ha\'in,2;  special  reference  to  Botanists.  Nae.i»:eli  und  Schwen- 
dener,  das  Mikroslvop.,  2  Aufl.  1877.  Dippel,  das  Milvi-oskop.,  2  Aufl. 
1882  Behren's  Helfsbuch,  etc.,  1883.  American  Edition,  Hervey. 
S.  E.  Cassiuo  &  Co.,  1885. 


LESSON  I. 

Use  of  the  Microscope.  Structure  of  Starch. 

The  separate  parts  of  the  Compound  Microscope,  as  seen 
in  the  Zeiss  stand  No.  vira  Fig.  2,  are  as  follows  :  the 
foot  fs;  the  column  sV  ;  the  stage  ot;  the  spring  sheath /"/(,• 
the  tube  t;  the  mirror  s,  concave  on  one  side  and  plane  on 
the  other ;  the  former  should  be  used  with  high  and  the 
latter  with  low  powers.  The  stage  has  a  circular  opening 
for  the  passage  of  the  light  from  the  mirror.  Beneath  this 
is  a  cylinder  diaphragm  c5,  fixed  in  a  slide  which  shoves 
into  the  stage  from  the  side. 

The  cylinder  diaphragms  have  openings  of  different 
sizes  and  are  movable  up  and  down  in  an  outer  cylinder, 
or  holder,  attached  to  the  slide.  It  should  be  first  barely 
inserted  in  the  holder,  and  after  that  has  been  pushed  into 
place,  it  should  then  be  raised  sufficiently  to  be  even  with 
the  top  of  the  stage.  The  amount  of  light  used  is  regulated 
by  the  diaphragm,  but  in  the  beginning  it  is  best  to  leave 
the  diaphragm  out.  Zeiss'  stands,  Nos.  \nb  and  viii,  have 
for  diaphragms  hemispherically-shaped  disks,  eccentrically 
pivoted  and  provided  with  openings  of  various  sizes  which, 
by  revolving  the  disk,  successively  come  within  the  optical 
axis  of  the  microscope.  *  The  stage  is  provided  with  remov- 
able spring  clips,/«:?,  for  holding  the  object-slide  in  place. 
The  tube,  t,  is  movable  in  the  spring  sheath  fli.  In  the 
larger  stands  the  tube  is  moved  with  a  rack  and  pinion, 
without  the  sheath. 

*  Ameiican  Microscopes  have  also  cliaphvagms  of  flat  disks  thus  perforated  and 
mounted.— A.  B.  H. 

(6) 


STRUCTURE    OF   STARCH. 


Fig.  2.  Stand  Vila  of  Zeiss  with  drawing  prism  |  natural  size.  fs.  foot;  sV,  un- 
der, and  si",  upper  part  of  column ;  ot,  stage;  eb,  cylinder  diaphragm  ;  fd,  spring- 
clips  ;s,  mirror;  n»,  line  adjustment  screw;  /Ä,  spring-sheath;  t,  tulie;  ot,  objective; 
oc,  ocular. 

Into  the  lower  end  of  the  tube,  screw  a  hnv-power  ob- 
jective and  set  one  of  the  weaker  ociihirs,  without  the 
camera  liicida,  cl,  into  the  upper  end.  Placing  the  mi- 
croscope before  a  window  and  looking  into  the  ocular,  ad- 


8  USE    OF    THE    MICROSCOPE. 

just  the  mirror  so  that  the  field  of  view  in  the  microscope 
will  be  brightly  and  iiniformlj  illuminated.  For  direct  il- 
lumination place  the  mirror  in  the  optical  axis  of  the  micro- 
scope as  it  is  not  ill  the  illustration.  The  quantity  of  light 
may  be  regulated  by  moving  the  mirror  up  or  down  upon 
its  carrier  within  the  optical  axis. 

Upon  a  clean  object-slide  put  a  small  drop  of  pure  water. 
Cut  a  potato  in  two  Avith  a  pocket  knife  and  put  some  of 
the  liquid  which  exudes  from  the  cut  surface  into  the  water 
drop,  and  place  over  it  a  clean  cover-glass. 

The  cover-glass  may  l)e  cleaned  bvholdin«:  and  rubbins: 
it  flatwise  between  the  fingers  with  a  piece  of  old  linen. 
If  the  drop  of  water  should  be  too  large  it  w^ill  run  out 
beyond  the  edge  of  the  cover-glass,  in  which  case  the  su- 
perfluous water  may  be  taken  up  with  a  piece  of  blotting 
paper  or  cloth.  It  is  better,  however,  to  make  a  new  prep- 
aration ;  for  the  action  of  the  blotting  paper,  in  soaking 
up  the  superfluous  water,  will  draw  out  a  great  part  of  the 
starch  grains  from  under  the  cover-glass. 

Place  the  preparation  on  the  stage  directly  over  the  open- 
ing. Push  the  tube  down  till  the  objective  nearly  touches 
the  object,  guiding  the  movement  with  the  e3'e  looking 
across  the  fetage  from  the  side.  Now  look  into  the  ocular 
and  draw  the  tube  very  slowly  upwards  with  a  rotary 
motion.  Soon,  the  previously  invisible  objects  will  ap- 
pear in  the  form  of  small  grains.  If  they  do  not,  and  the 
lens  is  drawn  back  2  cm.  or  more  froiji  the  object,  we 
may  conclude  either  that  the  object  is  not  in  the  field 
of  view,  or  that  we  have  drawn  the  tube  back  so  quickly 
that  the  image  has  been  so  rapidly  formed  and  dissipated 
as  to  escape  observation.  But,  to  find  the  image,  do  not 
run  the  tube  down  in  search  of  it,  else  we  shall  be  in  dan- 
ger of  pushing  the  lens  down  upon  the  object,  crushing 
the  cover-glass,  ruining  the  object  and  soiling  the  lens. 


STRUCTURE    OF    STARCH.  V 

Rather  proceed  as  at  first,  only  drawing  the  tube  back 
more  slowly  and  carefully. 

If  the  object  is  not  really  in  the  field,  put  it  there  by 
moving  the  slide,  and  find  it.  Having  found  the  grains, 
the  more  exact  focussing  may  be  finished  by  means  of  the 
micrometer  screw  m,  Fig.  2.  This  should  be  turned  ex- 
perimentally either  way,  the  adjustment  being*perfect  only 
Avhen  the  image  appears  with  the  utmost  possible  distinct- 
ness. With  the  larger  stands  the  coarse  adjustment  is  done 
with  the  rack  and  pinion  and  not  with  the  hand. 

Having  made  sure  of  the  existence  of  the  small  grains 
on  the  slide  in  the  field  of  the  microscope,  we  notice  the 
distance  which  the  objective  is  from  the  object,  and  note 
this  as  a  guide  for  its  future  use;  then,  leaving  the  ob- 
ject on  the  stage,  we  replace  the  low-power  lens  with  a 
higher  power  but  not  with  an  immersion  lens.  Push  the 
tube  down  till  the  lens  almost  touches  the  cover-glass  and 
focus  as  before,  remembering  to  draw  the  tube  upwards 
all  the  more  slowly  the  higher  the  power  of  the  lens. 
Directly  the  grains  become  visible,  finish  the  focussing 
with  the  fine-adjustment  screw.  Notice  now  the  shorter 
distance  of  the  lens  from  the  object. 

The  real  investigation  now  begins.  If  the  two  e3'es 
are  equally  good,  tiie  beginner  would  do  well  to  accustom 
himself  to  observe  with  the  left  eye.  This  will  leave  the 
right  eye  free  to  be  used  in  drawing,  while  he  continues  to 
observe  with  the  left  one.  The  beginner  should  always 
keep  the  eye  not  in  use  open.  At  first  his  attention  will 
be  distracted  by  surrounding  objects,  but  he  will  soon  over- 
come this  difficulty  and  be  able  to  concentrate  his  whole 
attention  on  the  object  seen  in  the  microscope  to  the  ex- 
clusion of  all  others. 

We  easily  see  that  the  granules  in  the  field  of  view  are 
solid  and  distinctly  laminated.     They  are  starch  grains. 


10 


USE    OF    THE    MICROSCOPE. 


Move  the  slide  till  a  point  is  found  where  the  grains  lie  a 
little  separated  from  each  other,  and  select  for  particular 
examination  such  grains  as  show  the  most  distinct  lamina- 
tion. Every  movement  communicated  to  the  object  by  the 
hand  will  be,  apparently,  greatly  increased,  and  exactly 
reversed  in  the  field  of  the  microscope.     The  difficulties 


Fig.  3.    starch  grains  from  the  potato.    A.  simple  grain ;  B,  a  semi-compound 
grain;  C  and  D,  wliolly  compound  grains;  c,  nucleus.    X  ö40. 

arising  from  this  will  be  overcome  onlj^  by  pi-actice. 

Having  selected  a  favorable  2:rain  for  examination,  re- 
place  the  ocular  with  a  stronger  one.  The  light  will  be  di- 
minished but  the  image  should  remain  distinct.  Readjust 
the  miri-or  so  as  to  get  as  much  light  as  possible.  If, 
when  moving  or  adjusting  the  preparation,  we  find  the 
image  become  suddenh'  indistinct,  we  shall  probal)lv  find 
it  to  be  caused  by  the  fluid  from  the  preparation  coming 
in  contact  with  the  lower  lens  of  the  objective.  This 
might  easily  happen  if  too  much  fluid  were  used  and  so 
should  exude  around  the  edo;e  of  the  cover-ijlass.  The 
lens  must  be  wiped  dry  with  a  piece  of  clean  linen,  or 
preferably  with  a  newly-cut  piece  of  elder  pith. 


STRUCTURE    OF   STARCH.  11 

Potato  starch-grains  (1)  are  relatively  large  and  are  ec- 
centrically formed  :  i.  e.,  their  organic  middle  point  c,  Fig. 
3,  A,  is  not  in  the  geometric  centre  but  nearer  one  end 
of  the  grain.  The  layers  are  not  uniformly  distinct;  but 
between  those  strongly  marked  are  others  much  less  dis- 
tinct, as  they  are  also  towards  the  edge  of  the  grain. 
The  organic  nucleus  on  account  of  its  great  thinness  is  rose- 
colored.  It  is  most  distinctly  so  where  it  is  hollowed  out. 
It  then  appears  as  a  rosy  point,  or  mark,  star  or  cross, 
with  a  dark  outline.  The  layers  immediatel}'^  surrounding 
the  nucleus  are  concentrically  developed,  but  the  eccen- 
tricity soon  develops  and  the  layers  thin  out  towards  one 
end  in  such  a  way  as  to  make  the  grain  in  this  direction 
quite  wedge-shaped.  On  the  less  fully  developed  portion 
of  the  grain  Avhich  we  will  call  the  anterior  end,  the  lam- 
ination appears  more  indistinct  on  account  of  its  being  at  a 
less  distance  from  the  surface.  The  individual  grains  vary 
in  size,  form  and  distinctness  of  lamination.  Air  bubbles 
often  appear  in  the  fluid  used  in  the  preparation,  and  may 
be  known  by  their  small  round  bright  centre,  and  their 
broad  dark  outer  bejt,  the  latter  being  darker  towards  the 
centre,  and  gray,  interspersed  with  bright  rings,  towards 
the  circumference.  This  peculiar  appearance  arises  from  the 
refraction  and  dispersion  of  the  rays  that  come  through 
the  babble,  except  the  central  ones,  by  passing  from  the 
denser  to  the  rarer  medium. 

By  focussing  upon  the  lower  part  of  the  bubble  the  dis- 
tinctness and  brightness  of  the  middle  disk  is  increased 
but  its  size  is  diminished,  while  the  breadth  of  the  sur- 
rounding black  belt  is  increased.  In  focussing  upon  the  top 
of  the  air  bubble  the  central  disk  is  increased  in  size  but 
diminished  in  brightness  ;  gray  rings  of  various  shades 
gather  about  it  and  the  surrounding  border  becomes 
smaller. 


12  USE    OF   THE    MICROSCOPE. 

Having  chosen  a  beautifully  lamluated  grain,  the  ob- 
server should  proceed  to  draw  it.  Drawing  is  of  the  first 
importance  in  microscopical  investigation.  We  first  learn 
to  see  an  object  clearly  when  we  observe  it  with  that  con- 
centration of  attention  necessary  to  its  graphical  repro- 
duction. Drawing  guards  against  superficial  or  cursory 
observation  requires  a  substantial  and  thorough  study  of 
the  image  and,  more  than  anything  else,  sharpens  our 
observing  faculty.  The  beginner  should  first  draw  the  ob- 
ject at  free  hand.  If  he  has  not  already  the  skill  neces- 
sary to  this,  a  little  practice  will  give  it.  The  object 
should  not  be  drawn  too  small  even  when  it  seems  very 
minute.  A  correct  judgment  of  its  real  size  in  the  field 
of  the  microscope  is  not  easily  acquired,  hence  it  is  well 
to  draw  it  too  large  even,  that  the  details  of  the  observa- 
tion may  be  put  in.  Not  less  important  is  it  to  provide 
the  individual  parts  of  the  image  with  corresponding  desig- 
nations and  note  the  name  of  the  plant,  the  object,  and 
the  most  important  results  of  the  observation. 

By  cautiously  pressing  upon  one  edge  of  the  cover-glass 
with  a  needle,  the  starch  grains  are  set  rolling  and  we  see 
that  they  are  somewhat  flattened.  Lamination  is  scarcely 
discernible  in  the  smallest  grains. 

Together  with  the  simple  grains,  A,  Fig.  3,  are  half- 
compound  grains  also,  B,  Fig.  3.  These  contain  seldom 
more  than  two  oroanic  nuclei.  Each  nucleus  is  surrounded 
with  a  number  of  individual  layers,  both  together  by  sev- 
eral common  layers.  Frequently,  the  two  systems  are 
separated  by  a  cleft  Avhich  cuts  down  to  the  common  lay- 
ers, jB.     The  number  of  layers  of  each  kind  vary  much. 

The  wholly-compound  grains  which  are  more  common 
than  the  half-compound  consist  of  two,  C,  rarel}'  of  three, 
D,  very  seldom  of  more  than  three  granules.  Unlike  the 
others  they  have  no  common  layers.     The  granules  turn 


STRUCTURE    OF    STARCH. 


13 


their  elongated  and  most  fully-developed  sides  towards  each 
other,  and  the  division  line  between  the  granules  often 
widens  into  a  cleft  towards  the  inside. 

For  purposes  of  comparison  make  also  a  preparation 
from  air-dried  potato  starch.  Put  a  trace  of  the  starch  into 
a  drop  of  water  as  before.  Before  replacing  the  first  prep- 
aration with  tins  one  on  the  microscope  stage,  withdraw 
the  tube  somewhat  and  raise  the  lens  away 
from  the  object.  Put  the  first  preparation  in 
the  moist-chamber  marked  so  as  to  identify 
it  at  any  time  later  on.  The  moist-chamber 
consists  of  a  deep  plate  and  a  glass  bell.  On 
the  plate  stands  the  zinc  rack,  Fig.  1,  on 
which  the  preparation  is  laid.  Water  suffi- 
cient to  immerse  the  bottom  of  the  glass  bell 
is  poured  into  the  plate.  If  the  water  under 
the  cover-glass  is  partly  evaporated,  put  a 
drop  on  the  slide  at  the  edo-e  of  the  cover-    „      ,    „      ^ 

•■  Ö  Fig.  4.    Starch 


run  in  under.      When  we  grains  from  the 


glass  and  it  Avil 

hn  Til  i."  HI  cotyledon  of  the 

ave  tocussed  the  new  preparation,  we  nnd  ^^g^,^    phaseoius 

that  the  lamination    of  the  air-dried    starch  vtdguris.  x  54o. 
grains  is  at  least  as  distinct  as  that  of  the  fresh  ones. 


This 
preparation  should  also  be  put  in  the  moist-chaml)er. 

We  will  now  make  a  preparation  of  air-dried  bean  meal, 
Phaseoius  vulgaris,  in  water.  The  grains,  Fig.  4,  are 
circular  or  oval,  a  little  flattened,  of  a  definite  medium  size. 
The  lamination  is  very  uniform  and  distinct,  the  structure 
concentric,  the  nucleus  concave,  elongated  somewhat  in 
the  oval  forms,  and  from  which  radial  clefts  extend  out- 
ward nearly  to  the  edge  of  the  grain. 

If  a  preparation  be  made  with  glycerine  the  grains  will 
appear  smaller  than  in  water.  No  trace  of  the  lamination 
inner  cavity  or  clefts  can  be  seen,  these  appearances  being 
caused  by  the  water  which  somewhat  swells  the  grains. 

Make  a  preparation  of  East  India  arrow-root  starch,  the 


14 


USE    OF   THE    MICROSCO:fE. 


commercitil  article  Curcuma  leucorrhiza.  If  one  reallj^has 
the  genuine,  the  grains  will  show  a  very  eccentric  struct- 
ure, Fig.  5  A,  contracted  at  the  anterior  end,  very  flat, 
and  beautifully  and  regularly  laminated.  Ol  ten  a  number 
of  the  grains  attach  themselves  by  their  flat  sides  and  when 
seen  from  the  edge  look  not  unlike  a  roll  of  coins,  B.  The 
size  and  form  of  the  grains  do  not  vary  much. 

The  West  India  arrow-root  obtained  from  the  rhizoma 
of  the  Maranta,  mostly  from  Ma~ 
ranta  arundinacea,  is  readily 
found  in  the  market,  but  is  much 
less  intei'estin<2:  than  the  East  In- 
dia  arrow-root.  In  water  the 
grains  resemble  those  from  the 
potato,  only  being  ;i  little  less  dis- 
tinctly and    therefore    uniformly 

laminated  :    somewhat  rounder, 
A  B 

FIG.  5.  starch  grains  from  the  Smaller  aud  more  uniform  m  size. 

commercial  Rust  Uidia  arrowroot,  J,j  pjjice  of  the  UUcleUS  OllC  fiuds 
Curcuma    leucorrhiza.       A,   side  i    r^    •       ,  i        ,-  p  •  i 

view;  5,  view  of  the  edges  or  two  a  clett  lu  the  tomi  ot  a  Wide  open 

adhering  grains.  X  540.  -y" 

Wheat  starch  shows  the  hmiination  very  poorly.  Split 
a  kernel  of  Triticum  durum  in  two  with  a  pocket  knife 
and  scrape  off"  a  little  substance  from  the  newly-cut  sur- 
face into  a  drop  of  water.  The 
large  grains  are  circular,  flattened, 
disk-shaped  and  regularly  lamina- 
ted. Fig.  6,  A;  still  the  layers  are 
difficult  to  see.  But,  in  many 
grains  they  and  the  nucleus  may  be 

distinctly  recognized.  Small  gi'ains  large,  i?,  small  grains,  x  sw. 
also  with  distinct  rose-colored  nuclei,  but  without  lamina- 
tion, will  be  seen.  Fig.  6,  B.  In  many  preparations  the 
compound  grains  are  common  ;  but  in  most  are  not  found, 
the  component  granules  having  fallen  apart. 


A 

Fig.   fi.     Staj-ch  grains  from 
heat,    Triticum    chiruvi.      A, 


STRUCTURE    OF    STARCH.  15 

We  will  now  halve  an  oat  kernel,  Avejia  sativa,  and  put 
a  little  of  the  substance  in  water.  We  now  have  the  com- 
pound grain  in  its  greatest  perfection,  Fig.  7,  A.  The 
size  of  the  compound  grains  and  the  number  of  the  com- 
ponent granules  greatly  differ.  Fig.  7,  A,  represents  one 
of  medium  size.  The  individual  gran- 
ules  are  polygonal  and  are  separated  /*=7>^  ' 
by  a  clear  boundary  line.     We  also      M^^^  ^ 

find    smaller  ones  consisting  of  but 
two  or  three  granules,  and  numerous 

single  granules  made  by  breaking  up       ^^^  ,     ^,^^^   g,.,i„3 
the  grains.     Lamination  is  not  to  be    from  the  oi\t,Avena  sativa. 

,       ,  A,   conipouinl-    grain;    B, 

seen,  and   the   nuclei  are   but   rarely    component  pans  of  tbe 
discernible.  .    '«"^«-  X  ^^°- 

The  starch  grains  of  the  Euphorbia  have  a  peculiar  ap- 
pearance. Cut  a  piece  at  will  from  the  Euphorbia  lielio- 
scopia  and  dip  the  cut  surface  into  a  drop  of  water  on  the 
slide.  The  milk-sap  which  runs  out  will  mingle  with  the 
water  and  we  shall  see  in  it  small  isolated  rod-like  bodies, 
Fig.  8.  They  are  the  starch  grains 
^^       ,%>x  i"  question.     They  are  strongly  re- 

|P   ^3^    X  X       tractive;  lamination   is   only   imper- 
-   \  •  <^  "''^>'     fectly  hinted  at  in  the  most  favorable 

l]   v:^  \      -^C^  eases  ;   in  many  instances  a  longitud- 

tl    ^k        ^^  ^"^'  ^^^^  ''^  ^^  ^'®  ^®^"  '"  ^^^®  inside  of 

11*^  the   grain.     The   size   of    the    grain 

Fig.  8.    starch  grains  varies  and  many  appear  to  be  smaller 

from  the  milk-sap  of  Eu-  in  the  middle. 

pliorbiahelioscopia.  X  510.  ,  ,  •       i       t-i       t       t  .  ^ 

In  the  tropical  Aup/iorbice,  the 
starch  grains  are  much  more  interesting.  Make  a  prepa- 
ration in  the  same  manner  from  Euphorbia  sp>lendens,  a 
plant  often  found  in  greenhouses. 

The  starch  grains  look  like  bones,  Fig.  9.     They  some- 


16 


USE    OF   THE   MICROSCOPE. 


grains  from  the  milk- 
sap  of  JSuphorbia 
splendens.  A  is  partly 
enveloped  in  a  thia 
membrane.    X  S^O- 


times  show  lamination.  Sometimes  a  colorless  sac  appears 
on  the  lateral  surface  of  the  grain,  A,  the  walls  of  which 
however  arise  not  from  the  substance  of  the  starch  grain, 
but  from  the  adhering  plasma-mass. 

The  small  globules  of  the  milk-sap, 
Avhich  are  distributed  through  the  water, 
are  seen  to  be  in  constant,  rapid,  trem- 
bling motion.  It  is  the  so-called  Brownian 
molecular  movement,  and  is  not  an  indi- 
cation of  life,  l)ut  is  perhaps  caused  by 
currents  in  the  fluid  which  move  the  gran- 
starch  ules. 

Having  made  this  general  survey  of  the 
form  and  structure  of  starch  grains,  "we 
will  now  study  the  effects  of  reagents  upon 
them  under  the  microscope.  Take  the 
potato  starch  preparation  ;  and,  after  focussing  it,  apply 
a  drop  of  iodine  solution  to  the  edge  of  the  cover-glass 
(iodine  water,  iodine  alcohol,  or  potassium  iodide  of  io- 
dine). Be  very  careful  and  not  get  a  drop  on  the  cover- 
slass  or  the  lens.     If  we  do  we  must  clean  it  at  once. 

Select  a  place  not  too  far  from  the  edge  of  the  cover- 
glass,  where  the  solution  was  applied,  and  then  move  the 
slide  to  follow  the  progress  of  the  reaction.  Directly  the 
grains  will  change  to  a  bright  blue  and  then  rapidly  darken 
to  a  black  blue.  At  first  the  lamination  becomes  more 
distinct  but  rapidly  disappears  as  the  grain  grows  opaque. 
A  larger  quantity  of  potassium  iodide  of  iodine  at  last 
produces  a  dark  In-own  color  in  the  grains.  Dry  starch 
turns  brown  in  the  fumes  of  iodine,  but  water  added  rap- 
idly changes  it  to  blue.  Touching  the  opposite  edge  of  the 
cover-glass  with  blotting  paper  will  hapten  the  movement 
of  the  reasrent. 


STRUCTURE    OF    STARCH.  17 

The  blue  reaction  with  iodine  proves  that  tlie  rod-like 
grains  of  the  Euphorbia  are  really  starch,  notwithstanding 
their  strange  form  and  lack  of  lamination. 

Let  us  study  the  actifii  of  potassiiun  hydroxide  on  starch 
grains.  Use  the  potato-starch,  introduce  the  reagent  and 
watch  its  effect  as  before.  The  reaction  should  be  very 
gradual  if  possible.  We  shall  tirst  see  the  lamination 
most  distinctly,  and  then  it  will  rapidly  disappear  as  the 
grain  swells.  During  this  swelling  the  nucleus  is  greatly 
hollowed  out  and  the  walls  of  the  anterior  part  of  the 
grain  are  folded  into  the  cavity.  Gradually  the  grain  be- 
comes a  transparent  mass  with  quite  indistinct  outline. 

Finally,  we  should  study  the  effect  of  heat  on  the  grains 
of  starch.  Warm  the  preparation  over  a  flame  till  it 
reaches  a  temperature  of  70  C,  taking  care  that  the  water 
is  kept  in  it,  wdien  it  will  be  found  that  the  grains  are 
swollen  in  exactly  the  same  way  as  when  treated  with  po- 
tassium hydroxide. 

Before  putting  the  microscope  away,  clean  the  lenses  as 
already  directed,  and  rub  the  tube  and  the  inside  of  the 
sheath  with  a  cloth.  Cov^er  the  instrument  with  a  glass 
bell  w'hich  should  be  bound  under  the  edge  with  felt. 

XOTES.      • 
(1)  Compare  Xaegeli,  Die  Stärkekörner,  in  Pflanzeiiph3'siol.  Unter- 
suchungen Heft  2:  E.  Strasburger,  Bau  u.  AVachsthum  der  Zellhjiute, 
p.  107.     There  is  also  other  literature. 
2 


LESSON  ir. 

Gluten  Meal.  Fatty  Oils.  Making  Permanent  Prep- 
arations. Use  or  the  Slviple  Microscope. 

With  a  stout  pocket  knife  bisect  the  cotyledons  of  a 
ripe  pea,  Pisum  sativum.  Witli  a  sharp  razor  take  a 
thin  section  from  the  cut  surface.  In  cutting  sections 
with  a  razor  observe  :  1.  The  surface  to  be  cut  should  be 
moistened  with  the  fluid  in  which  the  section  is  to  be  ex- 
amined, water  or  glyceriue, — in  this  case  with  the  latter. 
2.  The  first  section  is  not  to  be  used  since  the  tissue  is 
more  or  less  torn  by  the  pocket  kuife.  3.  In  cutting  hard 
tissue  like  this,  very  small  sections  of  the  utmost  possible 
tenuity  should  be  made,  care  being  taken  not  to  cut  deep 
lest  the  edge  of  the  razor  be  nicked.  4.  Lay  the  edge 
of  the  razor  upon  the  prepared  surface  and  cut  fiom  the 
middle  outward  towards  the  side  of  the  object.  5.  In 
cutting,  draw  the  razor  in  the  direction  of  its  length  as 
well  as  push  it  forward.  Both  hands  may  be  supported 
and  steadied  against  the  breast  and  yet  have  a  sufficiently 
free  movement.  The  back  of  the  blade  should  rest  upon 
the  forefinger  of  the  hand  holding  the  object.  6.  So  small 
and  hard  an  object  as  the  half  of  a  pea,  and  one  so  diffi- 
cult to  hold  firmly  in  the  hand  should  be  held  in  a  small 
hand-vice.  7.  Be  not  content  with  a  sino-le  section  but 
make  a  considerable  number  and  then  select  the  best  for 
the  investigation. 

The  section  should  be  examined  in  strong  glycerine  or 
with  glycerine  and  one-third  distilled  water.  Pure  water 
will  not  do,  as  it  causes  the  appearance  of  disorganization 
in  the  fundamental  substances  of  the  cells.     Use  a  hair 

(18) 


SECTIONS    OF    SEEDS. 


19 


pencil  to  traiisfor  the  section  from  the  knife  to  the  slide. 
Press  the  pencil  clown  upon  it,  in  taking  it  from  the  knife, 
which  will  prevent  it  rolling  up,  iis  it  often  does  when 
seized  at  the  edge  with  the  forceps.  Liy  the  section  care- 
fully in  a  drop  of  the  liquid  on  the  slide  and  withdraw 
the  pencil,  turning  it  laterally  in  the  fingers  a  little  at  the 
same  time. 

To  turn  the  section  over  upon  the  slide,  press  the  pen- 
cil down  upcm  the  slide  so  that  it  will  touch  the  edge  of 
the  section  and  then  rotate  it  in  a  direction  away  from  the 
section.  This  will  draw  the  section  over  upon  the  upper 
side  of  the  pencil,  when  it  may  be  put  down  again  upon 
the  slide  the  other  side  up.     Wash  the  pencil  after  using. 

Put  the  section  of  the  pea  under  a  moderately  high 
magnifying  power.  The  tissue  seems  to  he  composed  of 
rounded  cells.  Fig.  10.  At  the  place  where  these  cells 
meet  is  a  triansular  in- 


tercellular space  filled 
with  air.  It  is  black 
like  the  edo:e  of  an  air 
bubble.  1'he  walls  of 
the  cells  arc  pretty 
thick.  In  each  of  the 
cells  are  large  starch 
grains,  also  small  eran- 
ules,  al,  lying  between. 
The  latter  are,  in  their 
turn,  embedded  in  a 
very  tine  granular  sub- 
stance,   p.       At  thin 


e 


./^'':- 


Fig.  10.    Section  of  cotyledon  of  the  pea. 
wj.cell  membrane;  i,  intercellular  space;  am, 
starch;    al,    aleuron  grains;   p,  funilamental 
.  .  substance;  n.  nucleus,  the  last  maile  visi))le 

places  in  the  section  the       only  by  staining.    X  240. 

starch  »rains  are  fallen  out  and  we  see  the  corresnondino- 
cavities  in  this  fine,  granular  mass.  The  small  grains  are 
gluten  meal,  aleuron,  or  proteid  grains  (1).     They  lie  in 


20  STAINING    THE    SECTIONS. 

the  fundamental  substance  of  the  cells.  Apply  an  iodine 
solution  to  the  section,  and  the  various  elements  will  as- 
sume their  characteristic  colors.  Lift  up  the  cover-glass 
and  put  a  drop  of  the  iodine  directly  on  the  section.  The 
starch  grains  are  colored  blue  or  violet ;  the  aleuron 
grains  and  the  fundamental  substance  yellow.  AA'ith  po- 
tassium iodide  of  iodine  the  color  of  the  latter  elements 
becomes  very  intense,  but  t'le  starch  grains  are  over  col- 
ored and  become  a  dark  brown.  Put  a  section  in  a  drop 
of  borax-carmine  solution,  and  in  a  short  time  the  funda- 
mental substance,  and  directly  also  the  aleuron  grains,  will 
be  colored  a  dark  red,  while  the  starch  grains  remain  col- 
orless. The  reaction -is  more  evident,  it'  we  replace  the 
solution  with  water  or  dilute  glycerine.  This  may  be 
done  by  drawing  out  the  cannine  by  means  of  blotting 
paper,  while  at  the  same  time  the  other  liquid  is  supplied 
at  the  other  side  of  the  cover-glass.  A  drop  of  Millon's 
reagent  causes  the  starch  grains  to  swell  up  ver^' large  and 
soon  to  become  unrecoonizable.  But  the  aleuron  and  fun- 
damental  substance  is  disorganized,  and  the  mass  is  col- 
ored a  brick  red,  after  a  while. 

If  now  we  lay  a  section  in  a  solution  of  methyl-green 
and  acetic  acid,  there  will  appear  in  a  short  time,  in  each 
cell,  between  the  other  elements,  a  green-blue  fleck  of 
somewhat  indefinite  outline.  This  is  the  nucleus,  n. 
The  other  substances  have  not  been  colored.  The  starch 
o-rains  are  a  little  swollen  and  show  radial  clefts.  The 
aleuron  grains  are  a  little  enlarged  and  appear  porous  or 
hollow.  The  methyl-gieen  acetic  acid  is  thus  a  specific 
nucleus-stain  for  the  case  in  hand.  The  cell  walls  have 
also  been  stained  a  beautifid,  bright  blue  color  and  are 
much  more  distinctly  seen,  as  are  also  the  intercellular 
spaces,  than  in  the  glj'cerine  preparation. 

Thus,    we  have  learned  to  recognize  albuminous  sub- 


SECTION    OF    WHEAT    GRAIN. 


21 


stnnces,  for  such  are  the  aleuron  o-iains  and  the  protoplasm 
(cell  plasma  and  nucleus),  by  the  yello\v-l)ro\vn  reaction 
of  iodine,  the  absorption  of  coh)i'ing  matter  and  the  brick- 
red  reaction  of  Alillon's  reagent.  We  shall  learn  by  and 
by  that  protoplasm  will  show  these  reactions  only  when  it 
is  dead.  The  reagents  in  this  case  have  killed  it.  The 
nucleus  shows  a  particularly  strong  affinity  for  coloring 
matter. 

For  our  second  example,  selecting  a  kernel  of  wheat, 
Triticum  vulgare,  we  firs^t  cut  the  kernel  aci'oss  in 
halves  and  make  one-half  fast  in  a  hand-vice.  Moisten 
with  glycerine  and 
cnt  the  section  quite  , 
to  the  outer  surface 
of  the  kernel.  The 
section  examined  in 
the  same  fluid  will 
appear  as  in  Fig.  11. 
Beneath  the  com- 
pressed dead  cells 
of  the  skin,  p,  which 
represents  the  shell 
of  fruits  and  seeds, 
lies  a  layer  of  quad- 
rangular cells  close-  y\g.  ll.  section  of  a  grnin  of  wheat,  Triticum 
Iv  Dacked  with  aleu-  '"'^^"''^-  ?-'.  outer,  <,  inner  seeil-coat;«^,  aleuron;  a»» 
starch-grains;  ?i,  nucleus.    X  -^0. 

ron  grains.    Next  to 

tliis  are  elongated,  less  uniform  cells  which  contain  slanli 
grains  of  all  sizes.  These  points  are  all  established  by 
means  of   the  i)roper   reagents. 

Selecting  a  good  section,  we  will  now  proceed  to  make 
a  permanent  preparation.  We  will  mount  our  tirstol)ject 
in  the  simplest  possible  way  and  one  suitable  to  the  pies- 


22 


PREPARING-MICROSCOPES . 


eilt  case,  viz. ,  in  glycerine  jelly.  Put  as  much  of  the  jell}^- 
like  substance  on  the  centre  of  the  slide  as  we  judge  will 
make  a  small  drop.     Then  warm,  the  slide  over  a  flame 


B'  — 


Fig.  12.  Smaller  preparing  microscope  and  hand  rest  of  Zeiss,  §  natural  size, 
oi,  stage;  rf,  doublet;  st,  movable  rod;  sr,  fine  adjustment  screw;  s,  minor;  P, 
back  of  hand-rest. 


slowly  till  the  jelly  is  quite  liquefied.  Put  the  section  in 
the  fluid  and,  having  warmed  a  cover-glass,  lay  it  on  over 
it, — not  exactly  horizontal,  but  to  avoid  air  bubbles, — lay 
the  edge  of  the  cover-glass  upon  the  slide,  let  it  down 


PREPARING-MICROSCOPES.  23 

gradually  and  gently  upon  the  fluid,  pressing  it  horizon- 
tal afterwards.  If  still,  there  are  air  bubbles,  warm  the 
preparation  to  fluidity  again  and  gently  lift  up  one  edge 
of  the  cover-glass  a  little.  They  will  usually  come  out; 
if  not,  they  must  be  allowed  to  stay.  Several  small  sec- 
tions may  be  mounted  under  one  cover-glass,  by  carefully 
distributing  them  about  in  the  drop.  If,  in  putting  on  the 
cover-glass,  they  should  get  displaced  and  overlie  each 
other,  warm  the  slide  again,  and  with  a  stiff  hair  thrust  in 
under  the  cover-glass  push  the  sections  into  place. 

Before  putting  on  the  cover-glass,  in  the  first  place  the 
preparation  should  be  examined  carefidly  under  suita])le 
magnification,  and,  if  any  particles  of  dust  or  dirt  are  in 
the  fluid,  they  should  be  carefully  removed  with  a  needle. 
For  this  purpose  the  simple  or  compound  mounting-mi- 
croscope is  necessar}^,  and  we  will  now  direct  our  atten- 
tion to  learning  the  use  of  that. 

Let  us  suppose  that  the  student  has  the  small  Zeiss 
preparing-microscope,  or  any  other  of  like  construction. 
Over  the  stage,  oi.  Fig.  1^,  is  an  horizontal  arm  carrying 
a  doublet,  d.  The  arm  is  attached  to  a  steel  rod,  st,  Avhich 
rotates  and  moves  up  and  down  within  a  sheath,  the  latter 
movement  giving  the  coarse  adjustment.  The  fine  adjust- 
ment is  secured  by  turning  the  screw,  .sr.  The  instru- 
ment is  mounted  on  a  sul)stantial  and  suitable  block  whose 
raised  ends,  p,  serve  as  a  support  to  the  hand  inmani[)ulat- 
ing  the  preparation.  The  instrument  carries  two  and 
sometimes  three  doul)lets,  with  a  magnifying  power  re- 
spectivel}'  of  fifteen,  thirty  and  sixty  diameters,  and  may 
also  with  advantage  be  furnished  with  magnifying  glasses 
having  powers  of  five  and  ten  diameters. 

The  large  preparing-microsco[)e  of  Zeiss,  or  another  of 
like  construction  consists  of  a  lens  system  Fig.  13,  I,  in 
which  three  achromatic  lenses  are  united  in  one  objective, 


24 


PRRPARING-MICROSCOPES. 


o5,  a  tube,  and  an  achromatic  concave  ocular,  oc.  For  low 
magnification  the  objective  may  be  used  alone  as  a  magni- 
fying glass,  by  removing  the  tube  and  the  ocular.     The 


Fig.  is.  Larger  i)iei>aiing-micioscope  of  Zeiss,  i  natural  size,  ot,  stage ;  p,  hand- 
rest;  sr,  rack  and  pinion;  I,  lens  system;  oft,  objective;  oc,  ocular;  or,  an  object 
slide  on  the  stage  under  the  spring  clips.  ♦ 

lenses  are  also  separable  and  Ave  may  use 'the  upper  alone, 
or  the  upper  two  or  all  three  together  ;  the  resulting  magni- 
fication being  fifteen,  twenty  and  thirty  diameters,  respect- 


MAKING    PERMANENT    PREPARATIONS.  25 

ively.  Focus  with  the  screw,  sr.  On  both  sides  of  the 
st.'ise,  ot,  are  rests,  p,  for  supporting  the  Hands  while  at 
work. 

In  order  to  use  the  compound,  as  a  preparing-micro- 
scope,  we  ninst  attach  the  erecting  prism  to  ocuhir  No.  2, 
or  replace  this  ocular  by  one  to  which  a  prism  is  attached 
or  one  may  use  the  erecting  ocuhir,  only  that  the>e  go 
only  with  stands  having  a  draw-tube.  One  can  indeed 
train  himself  to  work  with  the  compound  microscope  with- 
out the  erecting  apparatus,  but  it  is  a  m-itter  of  much  dif- 
ficulty as  all  the  motions  seem  to  be  reversed  in  the  field 
of  the  micioscope."  Hand- rests  are  also  necessary  in  any 
case. 

Whatever  microscope  we  use,  we  place  the  preparation, 
from  which  we  are  to  remove  the  foreign  sul)stances,  on 
the  stage.  After  arranging  the  mirror  and  focussing  the 
object,  we  take  a  needle  by  the  1  older  in  each  hand,  sup- 
porting the  hands  on  the  rests,  and  bring  the  needle  points 
both  at  the  same  time  into  the  field  of  vision.  We  shall 
soon  learn  how  to  make  the  necessarily  very  small  motions 
required,  and  shall  have  removed  all  foreign  substances 
from  our  preparation  with  our  needle  points.  Having 
again  warmed  our  jelly  we  put  on  the  cover-glass. 

Glycerine  jelly  i)reparations  require  no  cementing  and 
are  therefore  extremely  simple  to  niid^e,  and  since  most 
vegetal)le  objects  and  the  stains  used  upon  them  are  Avell 
preserved  in  this  medium,  it  may  be  commended  before 
all  other  methods. 

Each  permanent  preparation  should  be  labelled  with  the 
name  of  the  pLint,  the  object,  the  preserving  medium, 
the  principalstains  used  and  the  date. 

We  will  now  go  on,  and  first  cut  a  section,  as  before 
directed,  of  the  seed  of  the  whhe  lupine,  Lupinus  alba, 
or    of  some    related  species,  moistening  the  cut   surface 


26 


ALBUMEN    CRYSTALS. 


with  water.  Examined  in  this  liquid,  the  section  shows, 
in  the  cells,  rounded  aleuion  grains  and  vacuoles.  The 
grains  are  strongly  refractive,  angular,  and  towards  the  in- 
terior become  gradually  reticulated  and  granular.  They 
lie  close  together  and  fill  the  cell,  being  embedded  in 
a  small  quantity  of  fundamental  substance,  which  also 
lines  the  cell  walls.  The  latter  are  thickened  and  dotted, 
a  structure  which  we  shall  study  further,  with  more  favor- 
able objects.  The  grains  are  colored  a  beautiful  gold  yel- 
low with  iodide  glycerine. 

We  will  next  make  a  section  from  a  Ricinus  seed.     The 

tissue    of  the    endo- 
j5  sperm    readily   lends 

itself  to  section-mak- 
ing as  it  contains 
much  oil  and  does  not 
need  to  be  moistened. 
Examined  in  water, 
this  liquid  expels  the 
oil  from  the  funda- 
mental substance. 
The  grains  embedded 
in  this  fatty  sub- 
stance. Fig.  14,  A,  have  within  them  mostly  one  but 
sometimes  also  two  or  more  albumen  crystals,  and  in 
most,  also  a  single  round  body  which  is  an  inorganic  sub- 
stance (a  globoid),  a  combination  of  phosphoric  acid  with 
lime  and  magnesia.  By  the  prolonged  action  of  water,  the 
fundamental  substance  will  be  disorganized,  large  drops 
of  oil  will  collect  on  and  in  the  object,  and  on  the  glass, 
and  on  this  in  irregular  masses.  But  if  they  float  free  in 
the  water  they  are  globular.  If  we  focus  upon  the  optical 
transection  of  such  an  oil  globule  it  will  appear  to  be  a 
bright  gray  and  be  surrounded  with  a  slender  black  band. 


Fig.  It.  From  endosperm  ot  liicinus  communis. 
A.  cell  with  contents  nnder  water;  D,  single  aleu- 
ron  grains  in  olive  oil;  ^,  globoids;  /j,  albumen 
crystals.    X  ßtO. 


,  VEGETABLE    OILS.  27 

Kiiise  the  tube  and  the  small  dark  band  becomes  some- 
what bi'oader.  Lower  the  tube  and  the  band  disappears, 
the  disk  showing  itself  somewhat  more  brightly  bordered. 
The  drop  of  oil  exhibits  an  appearance  the  exact  ojiposite 
to  that  which  we  o])served  in  the  air  bubble.  The  air 
refracts  the  light  less,  the  oil  more  powerfully  than  the 
Avater,  hence  their  contrary  behavior. 

Adding  now  a  drop  of  absolute  alcohol  to  the  Ricinus 
section  in  water,  the  preparation  is  made  more  clear  and 
the  crystals  of  albumen  in  the  aleuron  grains  are  brought 
out  sharply  to  view.  They  become  so  very  distinct  that 
this  method  of  examining  them  is  highly  commended. 
They  are  crystals  of  the  tetrahedric  hemiedrys  of  the  reg- 
ular system  (2).  Prolonged  action  of  the  alcohol  dissolves 
the  Ricinus  oil  drops,  it  differing  from  other  fatty  oils  in 
being  missible  with  alcohol. 

If  now  we  put  a  section  of  the  Ricinus  seed  in  glacial 
acetic  acid  the  albumen  crystals  will  disappear  in  the 
alenron  grains,  the  latter  in  their  turn  increasing  consid- 
erably in  size.  The  globoids  also  become  larger  and  show 
themselves  very  distinctly  in  each  grain.  No  dro[)s  of 
oil  are  seen,  Ricinus  oil  mixing  with  acetic  acid,  unlike 
that  of  other  plants.  Alcohol  and  glacial  acetic  acid,  in- 
asmuch as  they  dissolve  the  essential  but  not  the  fatty  oils, 
make  a  good  test  for  distinguishing  these  substances  under 
the  microscope. 

Turpentine  among  the  essential  oils  dissolves  somewhat 
less  readily  in  these  reagents  than  do  the  others.  Chloro- 
form and  ether  dissolve  fatty  and  essential  oils  in  the  same 
manner. 

Add  a  drop  of  dilute  alcana  tincture  to  a  section  in 
water  and  the  fatty  masses  will  be  colored  a  red  brown. 
The  same  result  follows  with  essential  oils  and  resins. 

To  a  glycerine  preparation  of  i?^c^■?m5  seed ,  add  a  small 


28  SECTION   OF   BRAZIL-NUT. 

quantity  of  hoematoxylin  and  it  will  color  the  albumen 
crystals  a  beautiful  violet.  With  olive  oil  the  albumen 
crystals  are  not  visible.  The  Avhole  grain  appears  as  a 
strongly  refracting  rounded  body  in  one  end  of  which  the 
globoid  seems  to  form  a  vacuole,  Fig  14,  B.  If  the  sec- 
tion be  laid  in  a  one  per  cent  solution  of  perosmic  acid 
the  all)umen  crystals  come  out  beautifully.  They  slowly 
assume  a  brown  shade.  Both  essential  and  fatty  oils  are 
slowly  blackened  with  perosmic  acid. 

Albumen  crystals  of  extraordinary  beauty,  which  read- 
ily exhibit  all  the  reactions  of  albumen,  are  found  in  the 
endosperm  of  the  seed  of  Bn'lJiolletia  excelsa,  which  may 
be  bought  anywhere  under  the  name  of  Brazil-nut.  The 
section  is  easily  made.  Add  absolute  alcohol  to  an  aque- 
ous preparation  and  the  crystals  come  out  most  distinctly. 
The  alcohol  does  not  perceptibly  affect  the  fatty  oil.  The 
latter  is  unchanged  by  the  action  of  glacial  acetic  acid, 
but  the  albumen  crystals  soon  dissolve.  In  a  one  per  cent 
solution  of  perosmic  acid  the  crystals  are  very  distinct. 
The  crystals  are  so  large  that  they  may  be  recognized  by 
their  form  alone,  even  with  comparatively  small  mngniti- 
cation.  Globoids,  in  the  shape  of  irregular  masses  of 
rounded  forms,  are  found  lying  in  the  tissue  with  the  crys- 
tals. The  fundamental  substance  is  very  rich  in  oily  mat- 
ter and  gradually  become»  almost  black  with  the  one  per 
cent  osmic  acid.  The  granular  contents  of  the  aleuron 
grains  soon  turn  dark  while  the  crystals  are  slowly  col- 
ored 3^ellow.  The  crystals  are  optically  uniaxial,  hexag- 
onal rhombahedra-hemiedrich. 

Notes. 

(1).     See  Pfeffer,  Jahrb.  f.  wiss.  Bot.,  viii,  p.  429. 
(2).     Shimper,   Uiitei-s.   ii.  d.  Proteinkrystalle  d.  Pfl.  Iiiaug.    Di.ss. 
Strassburg,  1878. 


LESSON  III. 

Streaming  Motion  in   Protoplasm.      The    Nucleus. 

Drawing  with  the  Camera.     Determining 

THE  Magnification. 


Selecting  the  hairs  on  the  stamens  of  the  Tradescantia, 
as  a  most  favoraI)le  object,  we  will  now  study  the  appear- 
ances of  motion  in  the  living  protoplasm.      Tradef^cantia 
Virginica  and  the  species  nearest  rehited  to 
it  are  cultivated  in  every  botanic  garden  and 
bloom  from  May  to  hite  autunm.    Select  hairs 
from  a  just- opening  or  recently-opened  blos- 
som.    Tear  away  a  tuft  of  the  hairs  from  the 
flower  with   the    forceps  and   transfer    it  to 
water.     The  Avhole  filament  may  be  put  under 
the  cover-glass  if  the  anther  has  been  removed. 
In  that  case  bubbles  of  air  usually  get  entan- 
gled among  the  hairs  which  costs  much  trouble 
to  get  out.     It  may  be  done,  however,  by 
brushing  it  with  a  fine  pencil  while  it  is  still     \j^  i  \\    \ 
held  in  place.     Put  on  the  cover-glass  and  if     \  v   ■    :\/j 
the  air  has  been  removed  with  sutKcient  care       \.  jj    ,•■';/ 
the  hairs  have  not  been  iniured.     The  hairs         >z.~:''-s 
will  be  seen  to  consist  of  a  series  of  swollen      ^^^  ^^    q^^^ 
cask-shaped  cells  ioined  end  to  end,  and  sep-  from  staminate 

T    ,  T .     .    .  1 ,  ,  -1     hair   of  Trades- 

arated  b}'  division  walls  at  the  constricted  cantta virginica. 
places.  Each  cell,  Fig.  15,  shows  a  thin  com-  ^  ^^^" 
plete  layer  of  protoplasm  lining  the  cell  Avail  and  numerous 
streams  of  protoplasm,  of  various  dimensions  running 
through  the  interior.  Suspended  within  these  streams, 
and  enclosed  in  a  coherent  layer  of  plasma,  is  the  nucleus 

(29) 


30  STREAMING    MOTION    IN    PROTOPLASM. 

(somewhat  below  the  middle  in  the  accompanying  illus- 
tration). A  violet  colored  cell-sap  fills  the  interior  of  the 
cell,  covering  the  nucleus  and  penetrated  by  the  proto- 
plasm streams.  Protoplasm  consists  of  a  colorless  viscid 
substance  called  hyaloplasm  which  bears  numerous  minute 
granules,  the  microsomata.  There  are  also  larger  strongly 
refractive  bodies  which  appear  to  be  of  a  bluish  color  and 
which  we  will  call  leucoplasts  or  starch-builders.  If  we 
focus  on  the  protoplasmic  wall  layer,  we  shall  see  that  it 
does  not  move  as  a  whole,  but  that  fine  netlike  anastomos- 
ing streams  run  through  it.  The  movement  is  especially 
strong  in  the  strings  of  plasma"  whioh  penetrate  the  cell 
cavity.  These  streams  are  of  different  thicknesses,  anas- 
tomose Literally  quite  often,  and  show  a  prevailing  ten- 
dency to  meet  at  the  nucleus.  Most  of  the  streams  end  in 
the  plasma  layer  which  surrounds  that.  The  streaming 
movement  is  often  only  in  one  direction  ;  but  often  also  in  a 
very  slender  string  two  streams  are  seen  moving  in  oppo- 
site directions.  We  perceive  the  movement  by  the  motion 
of  the  micros(nnes  and  leucoplasts.  Prolonging  the  ob- 
servation, we  notice  that  the  strings  slowly  change  their 
thickness,  arrangement  and  configuration.  New  connect- 
ing branches  are  pushed  out,  old  ones  grow  thin,  snap 
asunder  and  are  withdrawn  into  others.  So  the  image 
constantly  changes.  The  nucleus  is  almost  globular,  in 
many  cases  oval  or  somewhat  flattened.  AVith  the  highest 
magnification  which  we  employ,  it  appears  finely  dotted, 
and  in  it  may  also  be  distinguished  some  larger  grains. 
In  some  cells  the  nucleus  seems  to  have  divided  itself  into 
two  Avhich  lie  close  together.  The  nucleus  is  towed  about 
by  the  plasma  strings  and  so  gradually  changes  its  })lace 
in  the  cell.  To  demonstrate  this,  make  a  rough  sketch  of 
the  cell  and  contents,  and  after  some  little  time  compare 
it  with  the  then  position  of  the  nucleus  and  the  streams. 


ABBE    CAMERA   LUCIDA. 


31 


To  make  this  sketch  at  all  valual)le  it  should  be  quite  ac- 
curate, and  hence  should  be  drawn  with  the  camera.  Let 
us  learn  how  this  is  done. 

In  Fig.  15^  is  given  an  ideal  section  of  the  Abbe  cam- 
era lucida,  which,  after  focussing  the  object,  should  be  at- 
tached to  the  ocular  and  fastened  by  the  screw  sr.  It  will 
perhaps  be  safest  to  take  the  ocular  out  of  the  tube  for 
this  purpose,  and  avoid  the  danger  of  pushing  the  object- 
ive down  upon  the  preparation  by  the  operation.  Then 
adjust  the  mirror  of  the  camera  as  in  the  illustration  in- 


FiG.  15^.  Abbe's  camera  Incida,  natural  size,  longitndinal  section.  The  rays 
follow  direction  of  the  lines.  O,  direction  to  the  eye;  S,  direction  to  the  drawing 
surface;  sr,  clamping  screw. 

dining  it  at  an  angle  of  45°.  Now,  we  shall  see  an  image 
of  surrounding  ol^jects  in  the  field  of  the  microscope,  when 
we  look  into  the  ocular  through  the  camera.  Place  a 
drawing  board  horizontally,  by  the  side  of  the  microscope 
under  the  mirror ;  on  this  lay  the  drawing  paper  and  on 
this  hold  a  peucil  point.  If  this  is  visible  in  the  field  of 
the  microscope  along  with  the  image  of  the  object,  the  iu- 
strument  is  properly  adjusted.  The  pencil  becomes  visible 
by  a  double  reflection,  first  from  the  mirror  and  a  second 
time  from  the  silvered  surface  of  a  small  prism  in  the  cam- 
era (see  illustration),  while  the  image  of  the  object  comes 
direct  to  the  eye  through  a  small  opening  in  this  silvered 


32  DRAWING   WITH    CAMERA    LUCIDA. 

surface.  If  the  drawing  board  is  not  in  clear  view  of  the 
observer,  the  pencil  point  will  be  indistinct  and  the  board 
shonld  be  raised  up,  seldom  lowered.  The  distinctness  of 
the  microscopic  image  on  the  drawing  surface  depends  upon 
the  relative  amount  of  light  on  each.  That  on  the  draw- 
ino-  surface  is  regulated  by  a  contrivance  of  smoked  glass 
attached  to  the  camera.*  Having  rightly  adjusted  the  in- 
strument  and  the  dlumination,  trace  the  outlines  of  the  ob- 
ject in  the  tield  with  the  pencil. 

The  second  camera  recommended  in  the  introduction, 
has  the  advantage  of  being  always  attached  to  the  micro- 
scope and  ready  for  use.  It  consists  of  two  prisms  in  one 
mounting,  inclined  to  each  other  at  a  certain  degree,  liays 
from  the  pencil  to  the  eye  are  brought  to  be  parallel  with 
those  from  the  object,  by  a  double  reflection  in  the  prisms. 
The  camera  is  brought  into  position  when  the  front  edge  of 
the  prism,  visil)le  through  a  hole  in  the  mounting,  is  di- 
rectly over  the  eye-lens  of  the  ocular  and  nearly  halves 
it.     It  should  also  be  placed  close  down  to  the  ocular. 

The  drawing  board  is  inclined,  and  will  be  at  a  [)roper 
ano-le  if  the  circumference  of  the  field  of  view  in  the  micro- 
scope  makes  a  perfect  circle  when  drawn  on  the  board.  If 
it  appears  as  an  ellipse  when  so  drawn,  the  board  must  be 
fixed  at  a  greater  or  less  inclination  till  the  field  is  a  per- 
fect circle  on  the  drawing  paper  as  in  the  microscope. 

The  same  result  may  l)e  reached  l)y  using  a  stage  microm- 
eter ruled  to  hundredths  of  a  millimeter.  Arrange  the 
camera  so  that  the  successive  rulings  will  be  traced  on 
the  board,  one  beyond  the  other,  using  a  considerably  high 
magnifying  power,  and  then  carefully  measure  the  distance 
between  them.     If    that  distance  increase   outward  the 


*Tlie  desired  balance  of  illumination  may  also  be  obtained  by  tbe  use  of  the 
diaphragm  on  tlie  microscope,  and  a  small  screen  whicli  shall  throw  a  shadow 
upon  the  drawing  board.- A.  B.  H. 


MEASURING   WITH   CAMERA   LUCIDA.  33 

board  lies  too  nearly  horizontal ;  if  it  diminish  it  is  too 
nearly  perpendicular  ;  if  neither,  it  is  just  right,  and  then 
it  will  be  found  to  be  inclined  at  an  angle  of  al)out  25^. 

The  imaije  which  Ave  have  thrown  down  on  our  draw- 
ing  board  will  enable  us  to  determine  the  magnifying 
power  of  our  combination  of  lenses.  We  know,  for  ex- 
ample, that  the  lines  are  really  .01  mm.  apart.  But  on 
the  drawing  surface  they  appear  by  careful  measurement 
to  be  2.4  mm.  apart.  Hence  the  magnification  equals 
two  hundred  and  forty  diameters. 

If  one  has  attained  sufficient  exactness  in  drawing  to 
be  able  to  produce  a  picture  of  equal  dimensions  to  that  of 
the  microscopic  image  with  its  given  magnification,  he  has 
only  to  measure  this  and  divide  the  amount  by  the  mag- 
nification to  get  the  exact  size  of  the  object.  This  method 
gives  in  the  simplest  way  such  exact  results  that  we  may 
adopt  it  exclusively  in  our  investigations.  In  the  example 
before  us  the  hair  cell  of  the  Tradescantia  measures  9  mm. 
in  breadth.  This  divided  hy  240,  the  magnification,  gives 
.0375  mm.  as  the  real  breadth  of  the  cell.* 

We  will  now  return  and  attempt  to  draw  the  hair  cell 
of  our  plant  by  means  of  the  image  thrown  down  from  the 
camera.  We  shall  have  to  regulate  the  illumination  of 
the  second  camera,  by  shadowing  the  drawing  board,  and 
properly  adjusting  the  mirror,  for  we    should    have  an 

*  What  seems  to  me  a  far  better  and  more  exact  way  is,  to  draw  all  objects 
which  we  wish  to  measure  by  means  of  the  camera,  in  either  of  the  following 
ways: 

1.  Have  the  drawing  board  fixed  at  a  given  standard  distance  of  25  cm.  from 
the  camera  and  so  make  all  drawings  at  tliis  distance.  Then  divide  tlie  various  di- 
mensions of  the  drawing  as  above  with  the  known  magnification  of  tlielens  combi- 
nation used.  To  save  calculations  one  may  have  scales  made  corresponding  to 
the  magnification  of  the  various  combinations  of  lenses  by  which  to  measure  the 
drawing  at  once. 

2.  Let  the  drawing  board  be  at  any  convenient  distance  from  the  camera,  draw 
the  object,  then  without  changing  the  relative  position  of  anything  replace  the  ob- 
ject witli  the  stage  micrometer  and  draw  the  scale  of  .01  mm.  along  the  edge  of  tlie 
paper.    By  this  the  various  dimensions  are  always  easily  determined.— A.  B.  U, 


34  STREAMING   MOTION    OF   PROTOPLASM. 

equal  illumination  on  the  two.  Draw  with  a  lead  pencil 
on  stiff  smooth  drawing  paper.  A  very  thin  solution  of 
gum  applied  to  the  finished  drawing  will  prevent  it  "rub- 
bing" and  getting  defaced. 

We  Avill  make  a  sketch  of  the  outline  of  the  whole,  of 
the  streams  of  plasma  and  of  the  nucleus,  and  after  about 
an  hour  compare  the  object  and  the  picture  and  see  if  thej 
'Still  coincide  with  each  other.  We  shall  find,  as  already 
said,  that  the  divisions  of  the  streams  are  different  and 
the  position  of  the  nucleus  has  changed. 

In  order  to  demonstrate  that  the  streaming  motion  in 
the  several  cells  is  quite  independent  in  each,  and  that  it 
is  not  infiuenced  by  the  cell  walls  we  will  observe  the  efiect 
on  the  filament,  of  an  application  of  a  neutral  denser  fluid 
like  a  concentrated  solution  of  sugar,  or  strong  glycerine. 
Allowing  a  drop  of  the  fluid  to  run  under  the  cover-glass 
and  drawing  out  from  the  other  side  a  portion  of  the  water 
Avith  a  piece  of  blotting  paper,  we  shall  soon  seethe  efiect 
upon  the  cells  of  the  hair.  The  denser  fluid  absorbs  some 
of  the  water  in  the  cell,  which  causes  a  corresponding 
contraction  of  the  protoplasmic  sac  and  draws  it  away 
from  the  cell  wall  at  certain  points.  This  contraction  of 
the  protoplasmic  body  under  the  influence  of  a  water-ab- 
sorbing fluid  is  called  plasmal}sis.  While  the  contraction 
is  not  too  strong  it  is  ol)served  that  the  streamino"  on  and 
from  the  places  withdrawn  from  the  cell  wall  still  con- 
tinues.    Soon,  however,  all  movement  in  the  cell  ceases. 

It  will,  however,  be  resumed  again  if  the  denser  fluid  be 
replaced  by  water,  and  this  may  be  accomplished  hy  draw- 
ing out  the  fluid  from  under  the  cover-glass  by  means  of 
blotting  paper,  at  the  same  time  that  water  is  allowed  to 
run  in  under  from  the  opposite  edge  of  the  glass.  T-lie 
protoplasmic  sac  will  then  again  be  distended  till  it  reaches 
and  rests  upon  the  cell  wall.   It  often  happens  that  when  the 


STREAMING  MOTION  OF  PROTOPLASM.        35 

protoplasm  is  withdrawn  from  the  cell  wall,  little  masses  of 
the  plasma  will  be  torn  away  from  the  cell  body  and  lie  as 
rounded  balls  on  the  walls  of  the  cell.  All  these,  however, 
are  taken  up  andal)Sorbed  into  the  mass  of  the  protoplasm, 
when  it  is  restored  to  its  normal  place  and  conditions. 

One  may  easily  demonstrate  that. during  the  above- 
mentioned  contraction  of  the  contents,  the  coloring  mat- 
ter is  not  dilfused  throusjli  the  living  protoplasm,  and  that 
the  coloring  matter  of  the  cell-sap  is  correspondingly 
darker.  With  dead  cells  the  appearance  is  quite  differ- 
ent. The  application  of  absolute  alcohol  to  the  hairs  im- 
mediately kills  the  protoplasm  and  causes  it  to  absorb  the 
coloring  matter.  The  color  of  the  cell-sap  is  immediately 
withdrawn  and  it  becomes  very  clear,  while  the  cell  plasma 
and  the  nucleus  are  stained  a  dark  violet.  The  violet 
coloring  matter  can  now  penetrate  the  protoplasmic  sac 
and  distribute  itself  in  the  surrounding  fluid. 

In  lack  of  Tradescaiitia,  other  plant  hairs  may  be  sub- 
stituted, as,  for  example,  those  from  the  youngest  sprouts 
of  the  Cucurbita  species.  Cut  them  at  the  base  from  the 
plant,  with  a  razor,  and  transfer  to  a  drop  of  water  on  the 
slide.  The  stouter  hairs  are  composed  of  several  cells  at 
the  base,  but  change  into  a  pointed  row  of  cells  upward, 
while  others  bear  little  many-celled  knobs  on  their  points. 
The  network  of  protoplasm  is  richly  developed  in  the 
cells  and  contains  microsomes  and  larger,  less  numerous, 
green-colored  chlorophyll  grains.  The  nucleus  is  large, 
suspended  in  the  threads,  has  bright  nucleoli,  and  is  moved 
about  here  and  there  in  the  cell. 

The  root  hairs  of  the  Hydrocharis  morsus  ranee  alTord  a 
very  characteristic  ol)ject.  Take  the  young,  fresh  roots 
with  stiff  hairs  which  are  visible  to  the  naked  eye.  Cut 
off  the  end  of  the  root  and  lay  it  under  the  largest  cover- 
glass  in  a  sufficient  quantity  of  water.  On  account  of  the 
considerable  thickness  of  the  object,  all  parts  of  it  cannot 


36  ROTATION  OF  THE  PROTOPLASM. 

be  brought  within  the  focus  of  the  stronger  magnifications, 
the  lens  striking  the  cover-glass  before  the  deepest  parts  of 
the  object  are  in  focus.  The  hair  cells  are  very  long, 
sac-like.  All  root  hairs  are  single-celled.  The  rich  pro- 
toplasm is  in  powerful  motion ;  but  there  are  no  tine, 
net-like,  many -branched  streams,  only  a  single,  strong,  re- 
current wall-stream.  We  distinguish  this  kindof  stream- 
ino-from  the  other,  the  circulation,  by  naming  it  "rotation." 

This  stream  is  a  broad,  slightly  screw-like,  recurrent 
band  which,  if  projected  upon  a  plane,  would  form  a 
very  elongated  figure  8.  We  may  not,  perhaps,  represent 
the  movement,  as  if  the  band  as  a  whole  rotated  within 
the  cell,  since  we  observe  that  the  neighboring  particles 
continually  change  their  relative  position  during  the  move- 
ment. The  two  streams,  moving  in  opposite  directions,  do 
not  indeed  immediately  impinge  upon  each  other,  but  are 
separated  by  a  thin  layer  of  plasma  which  remains  at  rest. 

The  rotation  of  the  protoplasm  is  well  illustrated  in  the 
cells  of  the  leaf  of  Vallisneria  spiralis.  Make  a  section 
from  the  under  side  of  a  stout  leaf,  by  laying  the  long, 
slender  leaf  over  the  index  finger  of  the  left  hand,  hold- 
ino-  down  the  ends  with  the  thumb  and  third  finger,  and 
then  make  a  superficial  section,  with  a  razor,  of  about 
half  the  thickness  of  the  leaf,  and  lay  it  on  the  slide  with 
the  cut  surface  up.  Find  a  place  where  no  attached  air 
bubbles  interfere  with  the  observation,  and  then  selecting 
as  wide  and  long  a  cell  as  possible,  look  for  the  streaming 
movement.  The  movement  is  retarded  by  lowering  the 
temperature  and,  consequently,  accelerated  by  slightly 
warming  the  slide.  The  stream  circles  about  the  whole 
cell  without  essentially  deviating  from  a  direction  parallel 
to  its  longer  axis. 

The  "indifference  layer"  has  considerable  breadth.  The 
stream  carries  about  the  nucleus  and  the  green  chlorophyll 
grains.     The   former  is  flattened  disk-shaped.     It  is  for 


PROTOPLASMIC   STREAMING   IN   OHARA.  37 

the  most  part  hidden  by  the  chlorophyll  grains,  but  oc- 
casionally comes  in  sight.  Frequently,  it  gets  stuck 
fast  in  some  depression  or  turning  i)lace,  and  then  the 
chlorophyll  grains  get  dammed  up  against  it,  till  a  moment 
later  all  get  drawn  into  the  stream  again.  The  direction 
of  the  movement  chano;es  from  cell  to  cell  without  reo;a- 
larity.  By  adding  glycerine  or  sugur  solution  as  before, 
one  may  easily  see  the  movement  continue,  in  the  first 
moment  of  the  contraction  of  the  protoplasmic  sac. 

The  mostpowerful  plasma  stream  known  in  vegetable  cells 
is  met  with  in  the  Characeae.  We  must  use  the  genus 
JSfilella,  since  the  internodes  of  the  genus  CJiara  have  an 
opaque  outer  layer  which  renders  them  unserviceable  for  our 
purpose.  We  should  select  one  of  the  younger  internodes, 
and  we  shall  soon  observe  that  the  rotating  stratum  of  pro- 
toplasm has  a  very  consideral)le  thickness,  and  that  there  is 
an  outer  layer  in  which  are  embedded  the  chlorophyll  grains. 
This  layer  does  not  move.  It  is  in  this  case,  relatively, 
cpiite  thick,  but  is  commonly  so  thin  as  to  escape  observa- 
tion ;  for,  in  all  the  other  cases,  there  was  an  unmoving 
protoplasm-layer,  the  so-called  "skin  layer."  An  obliquely 
lying  stri[)e  on  the  wall  of  the  JSFitella,  easily  seen,  con- 
tains no  chlorophjdl  grains.  It  corresponds  to  the  "indif- 
ference layer"  of  the  protoplasm  stream.  It  repeats  here 
the  appearance  seen  in  the  root  hairs  of  HydrocJiaris 
when  the  "  indifference  stripe"  of  the  protoplasmic  layer 
is  likewise  found  extremely  reduced.  The  cells  of  the 
internodes  of  Chara  have  many  nuclei,  and  the  proto- 
plasm stream  bears  many'  elongated  nuclei  which,  only 
under  the  most  favorable  conditions,  are  discernil)le  as 
clear  spots.  The  rounded  masses  which  appear  in  the 
stream,  in  greater  or  less  number,  are  not  to  be  con- 
founded with  these.  They  have  either  a  smooth  surface 
or  are  covered  with  minute  spines.  Nothing  is  clearly 
known  of  their  sisrniticance. 


LESSON  IV. 
Chromatophores.     Colored  Cell-sap. 

"\Ye  have  alread}^  several  times  glanced  at  the  structure 

and  contents  of  the  chlorophyll  «[rains,  and  will  now  direct 

our  special  attention  to  these  forms.     For  this  purpose, 

we  shall  choose  a  widely  distributed  moss,  distinguished 

for  having  very  beautiful,  large,  lens-shaped  chlorophjdl 

grains,  and  whose  leaves,  constituted  of  a  single  layer  of 

cells,  are,  without   further   preparation,  m'ost   favorably 

adapted  to  our  purposes.     This  moss  is  Funaria  Jiygro- 

/-E.  ^       metrica.      Numerous    chlorophyll   graius    of 

'^"W      considerable  size  are  seen  in  each  cell ;  and,  in 

^lt:%^    plants  growing  in  diffused  daylight,  are  dis- 

^^"^0^      tributed  only  on  the  free  cell  walls,  that  is, 

(f^^       on  the  walls  which  constitute  the  upper  and 

under   surfaces    of  the  leaf.      They  present 
Fig.  16.   Ohio-         ,  ,  -^    ^ 

rophyii     grains  their  broad  side  to  the  observer.     That  they 

from  tlie  leaf  of  ii  i  •  xii  i 

Funaria  hygro-  '^^'®  Smaller  Avheu  secu  HI  proüic  w^e  observe 
metrica.  ji^  those  occasioual  instances  when  they  are 

found  lying  on  the  side  walls  of  the  cells.  Every  stage  in 
the  process  of  self-division  of  the  chlorophyll  grains  is 
easily  found,  often  in  one  cell.  (See  Fig.  16. )  The  resting 
grains  appear  almost  circular.  Then  they  Ijecome  ellipti- 
cal, then  biscuit-shaped  and  finally  completely  dissevered. 
The  two  young  grains  remain  for  a  lono-  time  in  contact. 
The  starch  contents  of  the  chlorophyll  grains  are,  accord- 
ing to  their  various  sizes,  in  many  leaves  easily  and  in 
others  with  difficulty  seen,  but  the  starch  comes  out  clearly 
if  the  chlorophyll  grain  is  set  free  in  the  water  and  disor- 
(38) 


CHLOROPHYLL   GRAINS.  39 

ganizecl.  For  this  purpose,  cut  the  leaf  into  small  pieces 
"with  sharp  shears,  and  the  freed  starch  from  the  chloro- 
ph^'ll  grains  will  increase  in  size  in  the  water  and  may  be 
easily  detected  with  iodine. 

An  uninjured  chlorophyll  grain  treated  with  iodine  is 
colored  brown,  this  being  the  result  of  the  combined  blue 
coloring  of  the  starch,  the  yellow  brown  of  the  protoplas- 
mic fundamental  substance,  and  the  green  of  the  chloro- 
phyll. Thebetter  way  is  to  bleach  a  leaf  by  long  immersion 
in  alcohol.  Then  the  iodine  solution,  gradually  penetrat- 
ing the  colorless  chlorophyll  grain,  will  color  the  starch 
within  before  it  does  the  protoplasmic  body.  The  iodine 
reaction  is  greatly  assisted  b}^  the  use  of  potash  which 
swells  the  starch  grains  and  thus  makes  the  least  possible 
quantity  of  starch  visible  in  the  chlorophyll  grains  (1). 
A  like  result  is  better  ol)tained  in  fresh  grains  by  treating 
them  on  the  slide  with  a  solution  of  five  parts  chloralhy- 
drate  in  two  parts  water  (  2 )  to  which  a  little  iodine  tincture 
is  added.  The  chlorophyll  is  dissolved  so  that  after  a  few 
minutes  the  leaf  becomes  colorless ;  at  the  same  time  the 
chlorophyll  grains  and  their  starch  contents  are  swollen 
and  in  the  latter  the  blue  cohn-  comes  out  distinctly.  Al- 
cohol-bleached leaves,  so  treated,  behave  in  the  same  way. 
If  treated  with  a  very  dilute  aqueous  solution  of  methyl 
violet  or  gentian  violet,  the  cell  membranes  are  stained, 
but  the  grains  still  more  and  become  more  distinct. 

The  living  chlorophyll  grains  of  the  Fnnaria  leaf  appear 
to  be  finely  dotted  under  a  high  magnifying  power  and  so 
betray  a  reticulated  structure. 

The  same  results  maybe  had  with  the  prothallium  of  the 
fern  so  that  either  object  may  replace  the  other.  The  pro- 
thallium  may  be  found  in  any  greenhouse  where  ferns  are 
cultivated.     Any  species  will  do  equally  well. 


40 


COLORED   ELEMENTS. 


For  the  study  of  other  colored  elements  (3)  we  will  take 
an  opening  blossom  of  Tropeolam  mcijus.  In  the  older 
blossoms  the  colored  bodies  are  beo-inninof  to  disoroftmize. 
AVith  the  forceps  thrust  into  the  tissue,  tear  off  a  piece 
from  the  upper  side  of  a  sepal.  Lay  the  strip  in  a  drop  of 
water  on  the  slide,  the  epidermis  up. 

Make  the  examination  at  once,  before  the  Avater  spoils 

the  colored  bodies,  and  select  an  uninjured  cell.       The 

colored  bodies  are  yellow  with  a  shade 

of  orange.       They    are   spindle-shaped 

three-  to  four-angled.     (See  Fig.  17.) 

The   uninjured  ones  are  homogeneous. 

^         AA'^ater  swells  them,    rounds   them  out 

\^;(       and     makes    vacuoles    or    water-filled 

spaces,  in  their  interior.     These  bodies 

are  especially  numerous  on  the  inside 

wall  of  the  epidermal  cells  of  the  upper 

side  of  the  calyx.     The  brown  stripe  on 

the  upper  side  of  the  sepals  arises  as  a 

section  would  show  from  the  red  cell-sap 

FIG  17.  From  the i,p-  which  fills  the  epidermal  cells.     These 

per  side  of  the  calyx  of  ^ 

Tropeoinm  mojus.  vn-  cclls   cOutaiu  also  ycUow  grains  which 

der  wall  of  an  epider-    ,1  i  i  11  i  ^  l   • 

mal    cell   with  color  the  colorcd  cell-sap  renders  almost  in- 


bodies  l3-in; 
510. 


on  it. 


X  visible.  The  nucleus  of  the  red  cells 
appears  mostly  as  a  clear  spot.  The 
petals  show  corresponding  relations.  The  edges  of  the 
lamella,  as  w^ell  as  the  cilia  at  their  base,  ma}^  be  used  for 
our  observation.  If  attached  air  bubbles  interfere  with 
the  examination,  a  slight  pressure  on  the  lamella  will 
drive  them  aw^ay.  But  the  sepals  are  to  be  preferred  to 
the  petals  for  examination  of  the  color-bodies,  on  account 
of  the  papilla  on  the  latter,  which,  by  their  own  form 
and  the  quantity  of  air  that  they  entangle  between  them, 


COLORED    CELL-SAP.  41 

materially  interfere  with  the  observation.  The  tier}"  red 
places,  at  the  base  of  the  petals,  arise  from  the  rosy  cell- 
sap  and  yellow  grains  in  the  epidermal  cells. 

During  the  examination^  it  has  been  observed  that  the 
upper  surface  of  the  epidermal  cells  of  the  upper  side  of 
the  sepals  are  longitudinally  striped.  The  stripes  pay  no 
regard  to  the  boundaries  of  the  several  cells,  and  are 
folds  of  the  cuticle  covering  the  epidermis. 

The  color-bodies  are  fairly  well  fixed  with  iodine  wa- 
ter and  are,  at  the  same  time,  colored  green  and  become 
very  distinct.  The  nucleus  is  stained  a  yellow-brown, 
and  its  nucleoli  are  distinctly  brought  out.  With  methyl 
or  gentian  violet,  the  color-bodies  are  stained  violet. 

Yellow  coloring  matter  is  almost  always  connected  with 
a  protoplasmic  substance,  but  we  sometimes  find  it  dis- 
solved in  the  cell-sap,  as  in  Verbascuvi  iiigrum.  Put  the 
petal  directly  in  the  drop  of  water  and  remove  the  at- 
tached air  as  much  as  possible. 

In  the  epidermal  cells,  on  either  side  of  the  leaf,  which 
have  a  wavy  outline,  the  yellow  cell-sap  is  seen.  The 
brown  spots  are  caused  by  purplish-brown  cell-sap.  In 
the  epidermis  of  the  stamens,  a  thin  lamella  of  wiiich 
may  be  taken  off  with  the  razor,  is  the  yellow  cell-sap ; 
also  an  irregular  mass  of  cinnabar-red  coloring  mutter,  and 
a  number  of  colorless  leucoplasts  filled  with  starch. 

In  the  under  lip  of  the  corolla,  A-niirrJiinuin  majus,  is 
a  sulphur-yellow  cell-sap.  The  red  portions  have,  in 
their  cells,  a  rosy  cell-sap  and,  partly,  also  one,  seldom 
more,  carmine-red  balls  of  coloring  matter. 

Rlue  cell-sap  may  be  found  in  the  epidermis  cells  of  the 
corolla  of  Vinca  major  or  minor.  The  epidermal  layer 
maybe  easily  torn  away  w^th  the  forceps.  The  side  walls 
of  the  cells  have  ledges  projecting  into  the  cell  cavity, 


i 


42  COLORED    CELL-SAP. 

Fig.  18,  which  are  swollen  at  their  inner  edge,  so  as  to 
become  T-shuped,  and  on  account  of  the  effect  of  unequal 
refraction  have  the  appearance  of  folds. 

Ros}^  ceü-sap  gives  the  color  to  the 

rose.     The  epidermis  may  be  easily 

torn  away.     It    is  deeply  papi Hated 

ir^  '^VV    ^^^'^  velvety.     The  cuticle  is  marked 

^^  ^(       by  distinct  stripes. 

j(^^  ^r  The  epidermis  of  both  sides  of  the 

^^^Xr^^^'^'^u-      "      bhie  sepals   of   Delphinum  consoUda 

FIG.  IS.    Epidermal  cell      cousisls    of    Cclls  with  WaVV    COUtour. 
of  the  linder  side  of   the 

petal  of  vinca  minor.  X  Oil  the  uppcr  sidc  the  ccll  riscs  into 
^*°'  a   papilla,   so    that   by   foTJUSsing   at 

about  half  its  heio'ht,  we  jjet  a  sun-like  figure.  The  cells 
contain  blue  cell-sap,  bordering  somewhat  on  the  violet. 
Besides  this,  many  cells  contain  blue  stars,  which  are 
produced  by  short  needles  crystallized  from  the  coloring 
matter.  The  epidermis  maybe  torn  away  in  small  pieces. 
But  the  sepal  is  transparent  enough  to  be  examined  through 
its  whole  thickness  at  the  edges,  after  removing  the  air. 

Examples  of  red  and  blue  cell-sap  may  almost  always 
be  met  with  in  red  and  blue  flowers,  among  the  more  strik- 
ing of  which,  is  the  intense  red-colored  flower  of  Adonis 
flamens.  Stripping  oft"  a  piece  of  the  epidermis  with  the 
forceps,  we  see  in  the  cells  beautiful  red,  nearly  round  to 
elliptical  grains,  nearly  as  large  as  chlorophyll  grains. 
They  appear  to  be  finely  granular,  and,  in  water,  dissolve 
into  very  fine  granules  which  exhibit  the  molecular  mo- 
tion. Tne  epidermal  cells  are  elongated,  the  cuticle 
longitudinally  striped,  the  stripes  running  distinctly  across 
the  boundaries  of  the  cells. 

.  The  orange-red  color  of  the  root  of  the  Daucus  carota 
arises  from  carmine  and  orange-red,  crystalline,  colored 


CRYSTALLINE    COLORED   BODIES.  43 

botlies;  their  commonest  form  is  represented  in  Fig.  19. 
they  are  small,  right-angled  rhombs,  often  elongated  to  a 
needle  shape,  again  prismatic  and  often  fan-shaped.  To 
these  crystalline  forms  are  often  attached  smallj  lateiidly- 
projecting,  starch  grains.  These  crystals  are,  therefore, 
original  sources  of  starch,  like  chlorophyll  grains  and  other 
colored  bodies  ;  but  the  cr3'stallized  coloring  matter  deter- 
mines the  form ;  the  crystals  have  bnt  a  very  small  quan- 
tity of  plasma  from  which  the  starch  originates. 

If  we  examine  the  variegated  varieties  of  trees  and 
shrubs,  or  even  of  the  herbaceous  plants  which  have  red- 
brown  leaves,  Ave  shall  tind  the 
cells  of  the  epidermis  filled  with  a 
rosy  cell-sap,  and  the  compound 
red-brown  color  is  the  eft'ect  of 
the  red  on  the  surface  and  the 
green  below. 

The  red,  autumn  color  of  the 
leaves  of  the  woodbine,  Amjpelopsis 
hederacea,  is  caused  by  the  rose- 
colored  cell-sap  in  the  cells  of  the 
tissue.      Distinct   yellow   autumn 

.  IT  Fig.  19.    Color-bodies  from 

colors  of  leaves  arise  from  the  dis-  the  root  of  carrot.  Part  with 
Organization  of  chlorophyll  grains,  ^t^rch  grains,  x  54o. 
as  is  most  beautifully  shown  in  the  leaves  of  GingTtO 
biloba,,  or,  lacking  these,  of  the  maple  species.  The 
brown  color  of  some  leaves  comes  from  the  correspond- 
ing color  of  the  cell  walls,  but  principally  of  the  cell  con- 
tents, as  may  be  easily  seen  in  the  oak. 

Starch  grains  originate  in  specially  individualized  pro- 
toplasmic forms,  as  in  chlorophyll  grains,  also  in  the  color 
substances,  where  starch  grains  may  often  be  detected, 
and,  tinally,  also  in  colorless  starch  generators  ;  the  latter 
assists  in  the  formation  of  starch  in  the  deep  layers  of  the 
plant  body.     We  may  name  all  these  together  chromato- 


44 


LEUCOPLASTS. 


phores,  and,  again,  the  chlorophyll  bodies  chloroplasts, 
the  colored  bodies  chromoplasts,  and  the  colorless  starch 
generators  leucnplasts.  These  forms  are  nearly  related 
and  may  be  transformed  into  one  another ;  they  all  belong 
to  the  protoplasm  of  the  cell  in  which  they  lie  embedded. 
On  the  contrary,  the  blue  stars  which  are  found  in  the 
cell-sap  of  DelpJdnum  consolida  do  not  belong  here,  but 
are  a  colored  substance  crystallized  from  the  cell-sap.  Like- 
wise the  colored  lumps  which  we  found  in  the  red  cell-sap 
of  Verbascum  is  not  a  chromatophore. 

The  largest  and  most  beautiful  starch  grains  are  pro- 
duced in  the  leucoplasts,  and  still  the  leucoplasts  are  not 
easily  seen  ;  a  relatively,  favorable  object  and  one  not  dif- 
ficult to  obtain,  for  this  purpose,  is  the 
rhizome  of /ns  Germanica.  Make  a  sec- 
tion parallel  to  the  surface  of  the  rhizome  ; 
directly,  the  onter-tissue  layer  is  re- 
moved, w^e  come  to  the  starch  layer;  ex- 
amine in  water.  In  nninjured  cells,  the 
leucoplasts  appear  as  collections  of  plasma 
on  the  posterior  end  of  the  starch  grains, 
Fig.  20.  The  latter  grow  here  only, 
and,  therefore,  have  an  eccentric  struct- 
ure. The  leucoplasts  become  granular  under  the  eye 
of  the  observer,  and,  finally,  dissolve  in  small  grains  and 
show  the  molecular  movement.  Two  starch  grains  are 
often  found  in  one  leucoplast ;  such  grains  soon  touch  each 
other  and  henceforward  have  layers  in  common  ;  these 
and  like  causes  produce  in  this  and  in  other  cases  com- 
pound starch  grains. 

NOTES. 

(1)  Methode  von  Böhm,  Sitzungsl)er.  d.  K.  A.  d.  W.  in  Wien,  Bd. 
XXII,  p.  479. 

(2)  Nach  A.  Meyer,  das  Chlorophyllkorn,  p.  28. 

(3)  A.  F.  W.  Shimper,  Bot.  Ztg.,  1880,  Sp.  881;  1881,  Sp.  185;  1883, 
105  und  Sp.  809 ;  A.  Meyer,  das  Chloropliyllkoru,  Bot.  Ztg.,  1883,  Sp.  489. 


Fig.  20.  Leuco- 
plasts with  starch- 
grains  from  root  of 
Iris  germanica.  X 
540. 


LESSON  V. 

Tissue,    Thickening  of  the  Wall,  Eeaction  on 
Sugar,  Inulin,  Nitrates,  Tannin,  Wood 
Substance  ok  Lignin. 

From  a  piece  of  the  white  sugar-beet  cut  a  section  par- 
allel to  the  lono-er  axis  and  in  the  direction  of  the  radius 
at  right  anijles  with  the  visible  rinojs  of  the  root.  Ex- 
amined  in  water,  it  will  be  seen  to  consist  of  nearly 
rio-ht-anoled  cells  tilled  with  a  watery  colorless  fluid.  The 
cell  walls  are  dotted  with  bright  round  or  oval  pits.  In 
occasional  cells  the  nucleus  is  visible.  The  intercellular 
spaces  are  mostly  tilled  with  air.  In  places  the  paren- 
chyma cells  become  slenderer,  are  elongated  lengthwise  of 
the  root  and  between  them  are  tubes,  mostly  air  filled, 
with  peculiar  thickening  of  the  walls.  These  tubes  are 
vessels.  Thickened  reticulated  ledges  cover  the  walls,  thin 
places  lying  between.  These  thin  places  or  pits  are  elon- 
gated transversely  to  the  length  of  the  vessel.  Ring-like 
thickenings  may  be  seen  now  and  then  projecting  from 
the  inside  of  the  vessels.  These  are  the  diaphragm-like 
■yemnants  of  originally  perfect  division  walls,  and  indicate 
that  the  vessels  originally  consisted  of  a  series  of  cells. 
The  air  may  be  drawn  from  the  vessels  by  means  of  an  air 
pump.  Lacking  this,  put  the  section  in  recently  boiled 
water,  or  better  still  in  alcohol.  This  liquid  will  kill  the 
cell  contents  but  that  does  not  matter  in  this  case. 

Occasionally,  we  shall  meet  with  a  cell  filled  with  small- 
klinorhombic  calcium  oxalate  crystals.  The  test  is  that 
they  do  not  dissolve  in  acetic  acid  but  do  in  sulphuric 

(45) 


46  STAINING  BEET    SECTIONS. 

acid.  Make  the  test  with  two  preparations.  The  result- 
ing gypsum  is  so  small  a  quantity  that  it  is  dissolved  in 
the  surrounding  liquid. 

Treat  the  section  with  an  aqueous  solution  of  methyl 
green  or  methyl  green  and  acetic  acid.  The  cell  wall  be- 
comes a  beautifid  green,  and  in  the  latter  case  the  cell 
contents  are  fixed  and  quickly  stained.  The  walls  of  cells 
and  vessels  are  colored  a  bluish  green.  Not  so  the  pits  on 
the  cell  walls,  which  are  the  thin  places  on  the  walls  of 
cells  not  otherwise  much  thickened.  Every  parenchyma 
cell  contains  a  nucleus,  having  a  distinct  nucleolus,  and 
surrounded  by  minute  leucloplast,  and  a  thin  layer  of 
protoplasm  on  the  wall.  The  vessels  have  neither.  To 
the  section  in  water  add  chloriodide  of  zinc  and  Ave  shall 
get  the  characteristic  violet  cellulose  reaction.  The  color- 
ing begins  on  the  edges  of  the  section,  but  it  may  require 
hours  to  become  complete.  The  vessels  are  colored  a 
brownish  yellow  like  lignitied  membrane.  The  pits  on 
the  cell  walls  are  uncolored  and  become  more  distinct. 
These  pitted  surfaces  are  always  oval  of  various  sizes  ir- 
regularly distributed  singly  or  in  groups.  The  larger  pit- 
ted places  are  overspread  with  violet  bands  of  different 
breadths,  making  the  appearance  of  fan-shaped  irregular 
lattice  work.  Bright  granules  colored  yellow  brown  by 
the  chloriodide  of  zinc  adhere  to  the  pitted  surface.  For 
comparison  produce  the  iodine  and  sulphuric  acid  cellulose 
reaction.  Impregnate  the  section  with  potassium  iodide  and 
transfer  to  dilute  sulphuric  acid  (two  parts  acid  and  one 
water).  It  will  be  colored  a  beautiful  blue.  The  smaller 
pitted  surfaces  are  still  uncolored,  the  larger  ones  latticed 
blue. 

Make  a  section  of  a  ripe  pear.  The  pulp  consists  of  reg- 
ular thin-walled  large  parenchyma  cells  somewhat  round- 
ed at  the  corners,  having  colorless  cell-sap,  a  much  reduced 


PEAR    STONES. 


47 


plasma  sac  and  a  nucleus.  Scattered  in  the  tissue  are 
nests  of  strongly  thickened  cells,  Fig.  21.  The  number 
of  these  united  stone  cells  is  different  in  different  places 
and  indifferent  species.  They  form  the  so-called  "stones" 
of  the  pear.  The  cells  are  distinguished  by  the  consid- 
erable thickening  of  their  walls  and  by  the  numerous,  fine 
branched  canals  penetrating  the  walls.  The  branching  is 
caused  I)y  a  number  of  the  canals  uniting  inwardly  as  the 
cell  cavity  becomes  narrower,  forming  a  common  canal 
which  opens  into  the  cell  cavity.  When  two  thickened 
cells  touch,  the  ca- 
nals meet.  In  the 
present  condition, 
the.se  cells  have  no 
liviiigcontents,  only 
a  watery  fluid.  Con- 
sequently they  rep- 
resent only  dead 
cell  husks.  Chlor- 
iodide  of  zinc  slowly 
colors  the  parenchy- 
ma cells  violet  and 
the  thickened  cells  a 
yellow  brown.  The 
latter  are  therefore 
lignified  and  on  ac-      ^,,.  „,    „       „       ,     ,  , 

°  ,  ,  Fig.  21.    From  the  pnip  of  the  pear.    Much  thick- 

COUnt  of  their  thick-    ened  «ells,  with  brandling  pore  canals.    .Siirrounaed 

IT       -r-  by  thin-walled  pai-encl)3-nia  cells.    V  210. 

ness  and    hgnihca-  i  .>  /\    *». 

tion  are  called  sclerenchyma  cells.  The  structure  of  thick- 
ened cells  is  well  brought  out  with  chloriodide  of  zinc. 
We  will  use  the  pulp  of  the  pear  for  our  microscopical 
study  of  the  sugar  reaction.  The  most  common  is  that 
with  Fehling's  solution  (34.64  g.  pure  crystallized  copper 
suli)hate,  200  g.  tartrate  of  potash  and  soda  dissolv^ed  in 
water) .     This  solution  may  be  kept  on  hand.     Take  about 


48  SUGAR  REACTIONS. 

600  ccm.  soda  lye,  sp.  gr.  1.12,  dilute  to  1000  ccm.  and 
boil.  The  section,  which  should  be  not  less  than  two  or 
three  layers  of  cells  in  thickness,  should  be  transferred 
from  the  Fehling's  solution  to  the  hot  lye,  and  the  reaction 
at  once  takes  place.  The  microscope  shows  in  the  cells 
the  vermilion-red  precipitate  of  reduced  cuprous  oxide. 
There  is  therefore  in  the  cells  of  the  pear  a  substance 
which  will  reduce  an  alkaline  cupric  oxide  solution,  a  sub- 
stance belonging-  to  the  grape  sugar  group  (glucose),  in 
this  case  grape  sugar. 

For  comparison,  make  the  investigation  with  a  section 
of  the  sugar  beet;  this  contains,  as  we  know,  cane  sugar. 
Immersing  the  section,  for  a  couple  of  seconds  in  the 
boiling  liquid,  gives  no  precipitate  in  the  cells;  the  sec- 
tion becomes  blue  ;  if  the  section  lies  for  a  long  time  in 
Fehling's  solution,  the  surface  begins  to  show  the  vermil- 
ion-red color.  The  cane  sugar  has  undergone  transfor- 
mation and  now  gives  the  cuprous  oxide  precipitate.  Under 
the  microscope,  vermilion-red  granules  appear  on  the  pe- 
ripheral cell  layer,  while,  if  the  reaction  has  not  continued 
too  long,  the  inner  cells  contain  a  blue  liquid. 

For  microscopical  purposes,  the  Barfoed  sugar  reaction 
with  acidulated  copper  acetate  has  much  to  commend  it. 
Dissolve  one  part  neutral,  crj^stallized  copper  acetate  in  fif- 
teen parts  water;  to  200  ccm.  of  this  solution  add  5  ccm. 
acetic  acid,  which  contains  thirty-eight  per  cent  glacial 
acetic  acid  ;  in  a  test-tube,  holding  from  5  to  8  ccm.  of  this 
solution,  put  a  not  too  thin  section  of  the  pear  and  in 
another  a  section  of  the  sugar  beet,  and  boil  up  for  a 
short  time  ;  pour  all  out  into  small  glass  dishes  and  let 
them  stand.  After  some  hours  we  shall  find  the  pear  sec- 
tion covered  with  a  fine  precipitate  of  cuprous  oxide  and 
a  small  quantity  also  of  the  precipitate  in  the  bottom  of 
the  dish,  while  the  beet-root  section  has  none  of  it.  The 
efiects  of  the  reaction  should  be  compared  after  a  few  hours, 


NITRITE    AND    NITRATK    REACTIONS. 


49 


since  a  small  quantity  of  the  precipitate  is  reoxidized  in 
the  air  after  a  longer  time  and  may  then  be  dissolved. 

We,  finally,  use  the  sugar  beet  to  observe  the  nitrate 
and  nitrite  reaction  by  means  of  diphenvlamin  (3).  This 
substance,  uSfed  by  the  chemist  as  a  most  delicate  test  for 
nitrates  and  nitrites,  is  very  useful  for  histological  re- 
search. jNIake  any  section  of  the  beet  which  shall  reach 
the  outer  surface,  lay  it  on  the  slide,  partly  dry  it  and 
add  the  reagent ;  this  consists  of  0.05  g.  diphenylamin, 
in  10  ccm.  pure  sulphuric  acid.  Immediately,  there  ap- 
pears in  the  outer  zone  of  the  section,  an  intense  blue 
color  ;  this  zone  contains 
the  latest  product  in  the  r,-;^  \ 
developing  tissue  of  the 
beet,  and,  consequently, 
is  that  part  which  con- 
tains the  nitrate.  Di- 
rectly, the  blue  color 
begins  to  spread  over  the 
rest  of  the  section,  but, 
at  first,  the  reaction  in 
the  colored  zone  is  quite 
sharply  defined.  We 
conclude  that  it  is  a  ni- 
trate and  not  a  nitrite, 
which  we  find  here,  because  the  former  is  much  oftener 
found  in  the  analysis  of  the  juices  of  the  plant.  We  partly 
dry  the  section,  so  that  the  reagent  and  the  color  will  not 
spread  so  rapidly  over  it,  and  the  colored  zone  will  be  more 
sharply  defined. 

Take    next  the   dahlia   bulb,  DaJdia   variabilis.     The 
longitudinally-halved  bulb  shows  the  central  pith:  a  lon- 
gitudinal section  of  this  shows,  under   the  microscope, 
many  series  of  nearly  rectangular  cells.  Fig.  22,  with  a 
4 


From  the  pith  of  Dalilia  variabilis. 


50 


IXULIN. 


much  reduced  protoplasmic  sac  with  nucleus  and  cell-sap, 
the  intercellular  spaces  being  filled  with  air,  and  the  cell 
walls  finely  striated,  the  striae  lying  at  an  angle  of  about 
35°  to  40°.  There  seem  to  be  two  opposite  systems  of 
stride  on  each  w^all ;  but,  in  fact,  they  belong  to  two  adja- 
cent walls,  and  the  extreme  tenuity  of  these  walls  enables 
us  to  see  the   two  systems  at  once.     The  cell  walls  are 

colored  violet  with  chlorio- 
dide  of  zinc,  but  when  the 
stripes  do  not  approach  each 
other  very  closely,  a  colorless 
line  is  seen  between  them. 
Like  the  pitted  surfaces  of  the 
wall,  these  unthickened  places 
are  not  colored  by  the  chlor- 
iodide  of  zinc.  Single,  rel- 
atively large,  rhombic-shaped 
places  come  out  with  great 
distinctness  as  pits  ;  these  pits 
ahvays  lie  on  the  dividing  line 
between  two  striae  and  at  the 
places  where  the  dividinglines 
of  one  system  cross  those  of 
the  other. 
FIG.  23.    From  the  bulb  of  7>«w*«       p^^^  ^  scction  lu   absolute 

variabihs  after  lying  in  alcohol  several 

months.    Sphere-crystals  on  the  walls,    alcohol    and  a  fine  precipitate 

^  ^*°'  of  inulin  will  be  produced  in 

the  cell-sap.  Eeplace  the  alcohol  with  water  and  warm 
the  slide,  and  the  precipitate  will  be  again  dissolved.  In 
order  to  study  the  spherical  crystals  which  the  inulin  forms, 
one  should  take  a  piece  of  the  bulb  which  has  been  in  al- 
cohol at  least  eight  days  ;  examine  the  section  in  water  and 
add  nitric  acid  very  slowly  at  the  same  time.  The  spherical 
crystals,  Fig.  23,  are  always  found  on  the  cell  walls;  they 


TANNIN   REACTION.  51 

form  more  or  less  perfect  globules  which  miiy  be  l)roken 
through  b}''  one  or  more  cell  walls.  For  the  most  part, 
globules  of  tlitterent  sizes  form  together  a  large  group ; 
the  globules  exhibit  more  or  less  clearly  a  radial  struct- 
ure, which  becomes  more  distiuct  after  the  uitric  acid 
begins  to  affect  them;  this  arises  from  the  radially  ar- 
ranged crystal  needles,  of  which  the  spherule  is  built. 
Besides  the  radial  structure,  a  concentric  lamination  is 
visible  which  is  understood  to  signify  an  unsteadiness  in 
the  conditions  of  crystallization.  Iodine  solutions  do  not 
color  these  spherules.  Warmed  in  a  drop  of  water  on  the 
slide  they  soon  disappear. 

There  is  nothing  better  than  a  gall  apple  for  testing  the 
tannin  reaction.  The  gall  apple  is  found  on  the  leaves  of 
the  oak  and  is  produced  by  the  sting  of  the  gall  wasp, 
which  thus  lays  an  egg  in  the  tissue  of  the  leaf.  Halve 
the  green  apple  and  make  a  delicate  radial  section.  The 
cavity  occupied  by  the  larva  is  surrounded  by  a  shell  which 
is  formed  of  isodiametric  oval  cells.  These  are  mostly 
richly  laden  with  starch  grains.  The  tissue  which  incloses 
this  consists  of  radially  elongated,  poh^gonal  cells,  which 
diminish  in  length  toward  the  periphery  of  the  apple,  and 
finally  end  among  the  small  cells,  towards  the  outside 
strongly  thickened,  outermost  cell  layer  of  the  epidermis. 
There  is  no  definitely  formed  cell-contents  in  all  this  tis- 
sue which  surrounds  the  inner  shell.  But  lay  a  freshly 
prepared  section  in  an  aqueous  solution  of  ferric  chloride 
or  ferric  sulphate,  and  the  whole  mass  Avill  be  colored  a 
deep  blue.  This  color  is  imparted  to  the  surrounding  liq- 
uid and  gives  us  the  iron-blue  reaction  of  tannin,  which 
may  also  have  an  iron-green  reaction.  Watching  the  re- 
action under  the  microscope,  by  putting  a  dry  section  under 
the  cover-glass,  and  then  adding  a  drop  of  the  iron  solu- 
tion, we  see  that  at  first  a  dark  blue  precipitate  is  thrown 


52  SCLERENCHTMA  TISSUE. 

down,  which  soon  dissolves  in  the  reagent  and  fills  the  cell 
with  the  blue  fluid.  The  starch-filled  cells  of  the  inner 
shell  give  the  weakest  tannin  reaction.  For  comparison 
Ave  will  lay  a  second  section  in  an  aqneous  ten  per  cent 
solution  of  potassium  bichromate,  and  we  shall  see-a  thick 
floccnlent,  reddish-brown,  permanent  precipitate  form  in 
the  cells.  We  shall  not  examine  the  vascular  bundles  of 
the  gall  apple  since  they  have  nothing  peculiar  to  do 
with  the  tannin  reaction. 

If  the  stem  of  a  st(mt  Vinca  major  be  cut  ofi"  near  the 
ground  and  then  broken  across,  we  shall  see  numerous 
small  fibres  projecting  from  the  edges  of  the  broken  parts. 
Seizing  them  with  the  forceps,  we  pull  them  out  and  put 
them  on  a  slide  in  a  drop  of  water.  We  shall  find  them 
to  be  long,  pointed,  much  thickened  sclerenchy ma  fibres. 
The  cell  cavity  is  reduced  to  a  narrow  tube,  and  towards 
the  ends  quite  obliterated.  In  the  less  thickened  walls 
we  find  but  one,  and  in  the  more  thickened,  two  systems 
of  striation,  the  one  belonging  to  the  inner  and  the  other 
to  the  outer  layer.  In  very  old  sclerenchyma  fibres,  often 
a  third  inner  system  is  seen  almost  perpendicular  to  the 
longer  axis  of  the  fibre.  The  latter  is  derived  from  retic- 
ulated  thickenings,  the  elongated  dots  appearing  between. 
These  innermost  thickening  systems  are  most  sharply  sep- 
arated from  the  outer.  With  chloriodide  of  zinc  the  fibres 
are  colored  a  violet  bordering  on  the  brown.  Especially 
instructive  is  the  behavior  of  cuprammonia  which  dissolves 
pure  cellulose.  The  efiect  must  be  quickly  and  closely 
Avatched.  The  reagent  greatly  swells  the  walls  of  the  fi- 
bres, at  first  making  the  striation  more  distinct,  but  quickly 
obliterating  it.  The  outer  layer  is  soon  perfectly  dissolved 
while  the  inner  reticulated  structure  longer  withstands 
the  action  of  the  reagent,  and  consequentl}'  becomes  at  last 
fully  isolated.     At  the  beginning  of  the  swelling,  a  still 


PITS    IN    CELL    WALL. 


53 


finer  lamination  shows  itself  in  the  already  visible  layers. 
So  each  layer  is  composed  of  a  number  of  extraordinarily 
fine  lamelloe.  This  very  fine  lamination  is  especially  well 
seen  in  the  inner  resisting  layer. 

Split  a  seed  of  the  Star-of-Bethlehem,  Ornithogalum  um- 
bellaium,  with  a  pocket  knife  and  make  a  very  thin  section, 
with  razor,  hand-vice  and  drop  of  water.  The  prepara- 
tion will  shoAV  nearly  right  angnlar  cells  as  in  Fig.  24. 
The  walls  are  much  thickened  but 
they  are  perforated  by  a  number  of 
simple  pits.  Looking  upon  the  sur- 
face of  the  wall,  these  pits  resemble 
round  pores,  m.  From  the  side  they 
reseml)le  canals,  running  from  the 
cell  cavity  to  the  primary  cell  wall. 
The  pits  of  neighboring  walls  meet 
and  are  separated  only  by  the  pri- 
mary cell  wall,^;,  which  we  call  here, 
the  closinsr  membrane.  The  inner 
surface  of  the  thickening  layer  is 
distinguished  by  its  stronger  refrac- 
tion, and  forms  the  boundary  mem- 
brane.      Add    sulphuric    acid    at   the    s\^erm  of  OrvWwr/alum  vmbel- 

laium.    m,  pit  from  above ;  j), 
edge    of    the     cover-glass,    an  I      the    closing  membrane  ;«,nucleHs! 

thickened  laj'er  will  be  dissolved,  ^  ^^^' 
while  a  network  of  very  delicate  walls  will  remain.  These 
walls  are  the  so-called  middle-lamella,  which  correspond 
to  the  original  walls  of  the  cell  before  the  beginning  of 
the  existing  thickening,  and  they  also  penetrate  the  clos- 
ing membrane  of  the  pits.  By  the  long  continued  action 
of  the  acid  they  too  would  disappear.  Chloriodide  of  zinc 
swells  the  thickened  layer  and  so  makes  the  middle  la- 
mella visible.  The  coloring  of  the  preparation  is  imper- 
fect in  consequence  of  the  swelling. 


Fig.   24.      From  the  endo- 


54  BORDERED   PITS. 

The  cells  are  closely  packed  with  protoplasm  and  granu- 
lar matter,  and  the  whole  contents  are  colored  yellow-brown 
with  iodine  solution.  The  nucleus  in  every  cell  may  be 
easily  demonstrated  by  means  of  acetic  methyl  green.  It 
generally  fails  in  no  living  cell  or  a  cell  capable  of  life. 

The  thickened  layer  of  the  cells  in  the  endosperm  of  the 
date,  PJicenix  dactyUfera,  has  a  similar  appearance.  But 
the  cells  are  elongated,  their  cell  cavity  narrower  and  their 
walls  thicker.  These  cells  are  in  the  date  germ  radially 
arransred.  Transverse  and  radial-lonijitiidinal  sections 
show  the  cells  in  longitudinal  section,  while  tangential 
sections,  perpendicular  to  the  radius,  show  the  cells  in 
transverse  section.  Chloriodide  of  zinc  colors  the  thick- 
ening layer  a  very  beautiful  violet,  and  a  prolonged  swell- 
ing causes  numerous  lamelUB  to  appear. 

We  will  now  turn  to  the  coniferous  wood  to  study  the  so- 
called  "  bordered  pits."  Take  a  piece  of  an  old  stem,  a  dry 
or  alcoholic  specimen,  and  with  a  sharp  knife  prepare  to 
make  the  diiferent  sections,  a  radial  parallel  to  the  longer 
axis,  a  tangential  also  longitudinal,  and  one  transverse  to 
this.  The  concentric  annual  rin2;s  of  the  wood  will  o-ive  us 
the  necessary  points  for  getting  the  desired  directions.  The 
radial-longitudinal  section  is  cut  perpendicular  to  these 
rings,  the  tangential  as  nearly  as  possible  parallel  to  them, 
the  transverse  perpendicular  to  both  the  others.  To  make 
good  wood  sections  and  not  spoil  the  razor  requires  the  ex- 
ercise of  the  greatest  caution.  In  case  the  razor  is  ground 
concave,  a  section  can  be  made  only  on  the  edges  of  the 
w^ood  or  only  so  far  as  that  the  back  of  the  razor  will  not 
strike  the  cutting  surface.  Still,  razors  used  to  cut  wood 
should  be  but  a  little  concave,  else  they  will  spring  and  cut 
unevenly.  The  best  form  is  that  which  is  ground  flat  on 
one  side,  but  this  has  the  fault  of  not  being  easily  sharp- 
ened.    The  cutting  surface  should  be  kept  moist,  the  sec- 


BORDERED   PITS    IX   CONIFEROUS   WOOD. 


55 


tion  mtide  as  thin  as  possible.  It  need  not  be  very  large. 
If  the  cutting  is  too  deep,  withdraw  the  knife  and  so  run 
no  risk  of  nicking  it.  The  razor  should  be  very  sharp  so 
as  not  to  mutilate  the  cell-membrane,  and  separate  the  in- 
ner thickened  layer  from  the  outer.  An  alcohol  specimen 
cuts  more  easily  than  the  dry  wood,  particuhirly  if  it  be 
subsequently  soaked  in  like  parts  of  glycerine  and  alcohol. 
The  first  section  cut  by  the  razor  should  not  be  used,  as 
the  cell  membranes  of  one  side  have  been  mutilated  b}^  the 
pocket  knife. 


Fig. -25.  Pinus  sylvestris.  A,  boi-flered  pit,  side  view;  B,  same  in  tangential  lon- 
gitudinal section;  t,  torus;  C,  transection  a  wliole  traclieid;  m,  middle  lamella; 
m*.  a  gusset  in  the  same;  i,  inner  cell  membrane.    X  510. 

With  a  low  power,  a  radial  section  is  seen  to  be  built  of 
longitudinally  elongated  cells  pointed  at  the  ends  and  at- 
tached to  each  other.  Crossing  over  these  cells  run  the 
cell  rows  of  the  medullary  rays  which  we  will  not  now 
consider.  We  focus  a  high  power  system  on  one  of  the 
broader  walls  of  the  longitudinally  elongated  wood  cells 
and  direct  our  whole  attention  to  the  bordered  pits  of  this 
wall.  The  bordered  pit  appears  to  us  in  the  form  of  two 
concentric  circles,  Fig.  25,  A.  The  inner  small  circle 
or  the  inner  ellipse  represents  only  the  opening  of  the 
pit  into  the  cell  cavity.     The  larger  outer  circle  is  the 


56  BORDERED    PITS. 

widest  pui't  of  the  pit  at  which  it  joins  the  primaiy  wall 
separating  the  two  cells.  This  bordered  pit  is  in  fact 
distinguished  from  the  simple  pit  as  we  have  seen  it  in  the 
Star-of-Bethlehem  and  in  the  date,  only  that  it  widens 
at  its  base.  The  pits  of  adjacent  cells  meet  in  this  as  in 
the  other  cases.  If  the  o[)ening  of  the  pit  (as  in  A)  is  an 
obliquely  placed  ellipse,  we  shall  also  find  by  changing  the 
focus  that  the  corresponding  pit  has  its  opening  inclined 
in  the  opposite  direction.  The  two  opposite  pit  cavities 
are  separated  from  each  other  by  the  primary  wall  which 
existed  before  the  secondaiv  thickening  had  be£>:un  and 
was  then  very  thin.  This  delicate  wall  is  the  closing 
membrane. 

This  is  thickened  in  the  middle  and  forms  the  so-called 
"torus."  By  careful  attention  and  focussing,  this  torus 
may  be  seen.  It  forms  a  smooth,  bright,  round  disk  of 
about  double  the  diameter  of  the  oritice  of  the  pit  (see 
in  A) .  In  favorable  cases  and  particularly  in  preparations 
from  dry  wood  this  thickening  of  the  membrane  is  seen  to 
possess  a  radial  striation  ;  so  that  it  would  seem  to  be 
diflerentiated  into  radially  running  lamellte  (6). 

We  shall  get  a  full  view  of  the  structure  of  the  bordered 
pit  only  by  means  of  a  tangential  section,  since  the  bor- 
dered pits  are  on  the  radial  walls  (7)  of  the  wood  cells 
and  will  be  cut  across  only  by  a  tangential  section.  See 
Fig.  25,  B. 

Look  for  the  pits  on  the  division  walls  of  the  widest 
wood  cells,  and  be  not  led  into  error  by  the  sections  of  the 
cells  of  the  medullary  rays.  The  image  of  the  dissected 
pit  will  be  seen  only  in  the  thinnest  and  most  delicate 
part  of  the  section.  There  they  will  appear  in  the  form 
of  two  o]i)en  pincer  jaws,  as  in  the  illustration  given  in 
Fig.  25,  B.  Kecognizing  the  structure  of  this  larger  pit, 
other  smaller  ones  will  be  easily  made  out.     The  thicker 


TRACHEiDES.  57 

the  Avail  the  longer  the  canal  and  the  wider  the  pit  cavity. 
In  the  most  favorable  eases  one  may  see  the  closing  mem- 
brane within  the  pit,  with  its  thickened  centre  t.  In  the 
larger  bordered  pits  it  is  mostly  pressed  over  to  one  side 
of  the  pit  cavity  and  seems  to  serve  the  purpose  of  a  valve . 
The  image  becomes  clearer  after  treating  the  section  to 
chloriodide  of  zinc  which  colors  the  cell  wall  a  yellow- 
brown.  Some  inner  layers  not  yet  fully  lignified  show^  a 
violet  tlush.  The  closing  membrane  is  not  colored.  This 
reagent  demonstrates  that  this  wood  cell  has  neither  nu- 
cleus nor  protoplasmic  sac.  It  consists  onl}'  of  dead  cell 
walls  and  resembles  the  vessels  in  its  function  of  conduct- 
ing water  as  well  as  in  the  manner  of  its  wall  thickening. 
It  is  called  a  tracheide  or  more  recently  a  hydroide. 

Often  the  coniferous  wood  which  we  are  examining  will 
be  seen  to  have  a  spiral  striation  with  an  ascent  of  about 
45°.  The  pit  openings  appear  thus  to  be  elongated  in 
the  direction  of  the  stritB  of  the  two  opposite  sides  of  the 
wall  on  which  they  are  placed  and  so  the  opposite  pit 
openings  seem  to  cross  each  other. 

AYe  will  now  make  a  very  delicate  transverse  section  of 
the  wood.  The  tracheides  are  prevailin2;lv  at  rioht  anales 
and  mostly  arranged  in  radial  rows.  On  the  radial  walls 
of  a  wide  cell  wc  find  the  pit  cut  in  section  Fig.  25,  C, 
whose  form  is  not  different  from  that  shown  in  the  tanaen- 
tial  section.  Between  the  cells  is  a  fine  dividing  line  m, 
the  middle  lamella.  AVhen  more  than  two  cells  meet  the 
middle  lamella  is  widened  into  a  solid  or  hollow  gusset  ??i*. 
The  inner  border  of  the  cell  w-all  is  more  refractive  and 
forms  the  boundary  membrane  i;  that  of  the  thick  walled 
tracheides  is  especially  distinct.  It  all  becomes  still 
clearer  by  the  use  of  concentrated  sulphuric  acid.  The 
thickened  layer  sw^ells  and  finally  dissolves  ;  the  boundary 
membrane,  withstanding   its  action    longest,   comes  out 


58  PHLOROGLUCIN   REACTION. 

sharply  to  view.  Between  the  swelling,  thickening  layers 
are  the  primary  walls  of  the  cells  and  these  at  last  remain 
a  delicate  network  colored  a  yellow-brown.  This  acid- 
resisting,  middle  lamella  is  "cutiiiized."  By  the  gradual 
swelling  of  the  thickening  layer  in  sulphuric  acid  we  find 
that  it  is  composed  of  numerous  extremely  delicate  lamel- 
lae. Chloriodide  of  zinc  colors  the  section  yellow-brown 
but  in  some  cases  the  innermost  part  of  the  thickening  layer 
takes  a  violet  tinge.  If  Ave  follow  the  chloriodide  of  zinc 
treatment  with  dilute  sulphuric  acid  (two  parts  acid  and 
one  of  water)  the  whole  thickening  layer  will  be  colored 
blue.  Treat  the  section  with  chromic  acid  and  the  middle 
lamella  will  be  dissolved  and  the  cells  separated.  The 
thickening  layer  will  swell  somewhat,  the  lining  membrane 
at  first  showing  up  sharply  and  afterwards  disappearing. 

Phloroglucin  and  aniline  sulphate  give  characteristic 
reactions  with  wood  substances  or  lignin  (8).  Dissolve 
a  small  portion  of  the  phloroglucin  in  alcohol  and  lay  the 
section  in  the  solution.  Afterwards  put  it  in  a  drop  of 
water  on  the  slide  and  add  a  little  hydrochloric  acid  at  the 
edge  of  the  cover-glass.  Directly  the  cell  walls  will  be 
stained  a  beautiful  violet  red  color.  An  aqueous  solution 
of  aniline  sulphate  colors  Avood  a  bright  yellow,  but  the 
color  is  heightened  by  adding  a  dilute  sulphuric  acid.  In 
place  of  the  phloroglucin,  one  may  use  an  aqueous  or  al- 
coholic extract  of  cherry  Avood,  Avith  almost  the  same  re- 
sults (9).  Treat  n  section  from  a  fresh  stem  of  fir  AVood 
Avhich  has  either  the  pith  cells  or  the  bark  cells,  with  con- 
centrated hydrochloric  acid.  Immediately  the  Avood  Avill 
be  colored  yellow,  Avhich  afterwards  gradually  softens  to 
a  violet  (10).  This  is  also  a  phloroglucin  reaction,,  the 
phloroglucin  being  derived  from  the  pith  or  bark  cells.  The 
medullary  rays  themselves  in  young  Avood  contain  some 
phloroglucin. 


REACTION    ON    LIGNIFIED    WALLS.  59 

Thus  the  tliflerent  behavior  of  lignifiod  and  unlignitied 
cell  walls  toward  coloring  matter  is  one  important  element 
in  their  investigation. 

Notes. 

(1)  Compare  Sachs,  finally  Jahrb.  f.  wiss.  Bot.,  Bd.  iii,  p.  187. 

(2)  Ban'oed  de  organiske  Stoffers  qualitative  analyse  Kjöbenhavn, 
1878,  pp.  210,  217,  223  Aum. 

(3)  See  H.  Molisch  :  Ber.  d.  deut.  bot.  Gesell.  I  Jahrg.  p.  150. 

(4)  Sachs,  Bot.  Zeitg.,  ISGi,  p.  77;  Hausen,  Arb.  d.  Bot.  Inst,  in 
Wurzburg,  Bd.  Ill,  p.  108;  Meyer,  Bot.  Ztg.  1883,  Sp.  334. 

(5)  Sauio,  Jahrb.  f.  wiss.  Bot.  Bd.  ix,  p.  50;  S'trasburger,  Zell- 
häute, p.  38;  Eussow,  Bet.  Centralbl.  Bd.  xiii,  No.  1-5.  There  is  also 
other  literature. 

(6)  See  Eussow,  Bot.  Centralbl.,  1883,  Bd.  xiii,  No.  1-5. 

(7)  Taugeutially  placed,  bordered  pits,  which  are  so  rare  in  the  fir, 
quite  i-eguhuly  occur  in  those  wood  cells  of  other  Abietince,  which  are 
formed  in  the  autumn. 

(8)  Both  introduced  by  Wiesner  (See  Stzber.  d.  Math.  nat.  Kl.  d. 
Akad.  d.  Wiss.  Bd.  lxxvii,  1  Abth.  und  früher  schon  a.  a.  O.) 

(D)  V.  Höhnel,  Sitzber.  d.  Math.  n.  Kl.  d.  Wiener,  Akad.  d.  Wiss. 
Bd.  Lxxvi,  p.  685. 

(10)     The  same,  p.  676. 


LESSON  VI. 

EriDERMis.     Stomata. 

Prepare  a  superficial  section  from  the  outside  (under- 
side) of  the  "riding"  leaf  oi  Iris  florenlina.  The  section 
should  be  so  thin  that  but  traces  of  the  underlying  tissue 
should  adhere  to  the  epidermis ;  examine  in  water,  the 
outside  uppermost.  The  epidermis  consists  of  much  elon- 
gated cells  running  parallel  to  the  axis  of  the  leaf.  The 
cells  end  in  a  transverse  division  wall,  are  joined  together 
without  intercellular  spaces,  contain  colorless  cell-sap,  a 
reduced  plasma  sac  and  a  nucleus.  The  outside  of  the 
epidermis  is  covered  with  an  extraordinarily,  fine-grained 
wax.  On  a  line  with  the  epidermal  cells  lie  the  elliptical 
stomata  indistinctly  seen.  The  four  contiguous  surface 
cells  reach  over  and  partly  cover  the  guard  cells  of  the 
stoma,  so  there  remains  only  an  elongated,  elliptical,  mi- 
nute cavity,  f,  which  leads  to  the  stoma.  Fig.  26,  A;  this 
cavity  is  filled  with  air  and  appears  mostly  black.  Turn 
the  section  over  now,  and  it  will  easily  be  seen  that  the 
stoma  is  formed  of  two  semilunar  shaped  cells  ;  unlike 
the  neighboring  cells  of  the  cuticle,  they  contain  chloro- 
ph^dl  grains.  The  nucleus  is  seen  as  a  clear  spot,  usually 
for  half  the  length  of  the  cell.  Between  the  two  guard- 
cells  occurs  a  spindle-shaped  opening,  s,  which  extends 
along  half  the  length  of  the  cells.  Make  a  section  now, 
crosswise  of  the  leaf,  and  you  will  naturally  make  it  trans- 
versely across  the  stomata.  For  this  purpose,  cut  out  a 
small  piece  from  the  leaf  and  make  the  section,  while 
holding  it  between  the  two  halves  of  an  elder  pith.  A 
(60) 


STOMATA. 


61 


piece  of  the  pith,  3  cm.  long,  may  be  carefully  split  in 
two,  lengthwise,  the  piece  of  leaf  laid  between  the  two 
halves  in  such  a  way  as  to  bring  the  edge  to  be  cut  just 
above  the  end  of  the  pith  ;  hold  the  pith  in  the  fingers  or 
put  a  light  rubber  band  around  it  to  hold  it  in  place,  or 
even  fasten  it  in  the  hand-vice,  but  hold  it  so  that  the 
knife  will  cut  along  the  bioad  surftice  of  the  object  and  not 


Fig.  26.  Epklei-niis of  the  underside  of  the  leaf  of  Iris  floreniina.  ^,  surface 
view;  B,  transection;/,  minute  depression ;  s,  stoma;  c,  cuticle;  a,  breathing  cavity. 
X  210. 

upon  the  edge.  For  very  delicate  objects,  the  sunflower 
pith  is  better  than  elder  pith.  Cut  first  both  the  end  of 
the  pith  and  of  the  object,  quite  clean  away,  then  cutting 
through  both  at  the  same  time,  make  several  very  thin 
sections,  keeping  the  cutting  surface  nioistened  and  a  drop 
of  water  on  the  razor  blade.  Remove  the  sections  from 
the  knife  to  a  slide  by  means  of  a  hair  pencil.    The  prep- 


62  LEAF   SECTIONS.     ST03[ATA. 

aration  of  sufficiently  delicate  sections  need  occasion  no 
great  difficulty,  but  if  such  a  difficulty  arise,  it  ma}^  be 
met  with  a  microtome ;  a  hand  microtome  of  the  simplest 
construction  will  be  sufficient.  The  pith  holding  the  ob- 
ject may  be  fitted  into  a  cork  and  this  into  the  well  of  the 
microtome,  the  pith  rising  some  little  above  the  •  cork. 
The  section  nui}^  be  made  Avith  a  common  or  a  flat-sided 
razor,  cutting  free-hand  across  the  top  on  the  brass  or 
glass  j)late,  and  lifting  the  object  each  time  the  required 
distances  by  means  of  the  screvv.  More  elaborate  micro- 
tomes, useful  to  the  zoologist,  are  superfluous  to  the  bota- 
nist.* 

jNlake  a  number  of  sections  and  put  them  in  a  watch 
glass.  Examining  a  section  in  water,  we  find  a  stoma  cut 
throuah  the  middle,  as  in  Fig.  26,  B.  First,  we  notice 
that  the  cells  of  the  cuticle  are  thicker-walled  without 
than  within.  Still  the  inner  walls  are  pretty  thick,  while 
the  radial  walls  are  very  thin  ;  these  are  connected  with 
the  function  of  the  cuticle  which  is  not  only  to  protect  the 
leaf  without,  but  also  to  be  a  water  reservoir  (2).  The 
thin  radial  walls  are  adapted  to  an  easy  change  in  the  vol- 
ume of  the  cells,  for,  acting  like  a  bellows,  they  diminish 
their  height  with  the  loss  of  crater,  and,  again,  increase  it 
when  the  water  is  increased.  The  guard-cells  lie  deep 
beneath  and  between  the  cells  of  the  cuticle.  The  little 
pit,  /",  leads  down  to  the  guard-cells ;  these  are  much 
thickened  above  and  below.  These  thickened  sides  jut 
out  towards  each  other  in  the  opening ;  al)ove  these  thick- 
ened places  are  peculiar,  beak-shaped  projections.  On 
the  opposite  side  towards  the  inside  of  the  cells  of  the 
cuticle,  the  walls  of  the  guard-cells  are  relatively  quite 
thin.      On  this  method  of   wall  thickening  depends   the 

♦Taylor's  freezing  microtome  is  very  serviceable  for  cutting  soft  tissue  con- 
taining water. — A.  B.  H. 


GUARD-CELLS    OF    STOMATA.  63 

mechanism  for  the  movement  of  the  guard-cells  Avhich  are 
more  curved  and  the  orifice  opened,  the  greater  their 
turgidity ;  and,  conversely,  are  more  extended  and  the 
orifice  closed  or  narrowed  b}' a  decrease  of  their  turgidity. 
It  is,  in  fact,  clear  that  the  guard-cells,  by  increased  tur- 
gidity, become  more  convex  on  the  side  of  least  resist- 
ance and  concave  on  the  side  of  most  resistance,  like  a 
rubber  bag  with  a  thicker  wall  on  one  side  than  on  the 
other.  If  water  or  air  be  forced  into  it  under  high  pres- 
sure, the  side  making  the  strongest  resistance  becomes 
concave,  while  that  making  the  least  becomes  more  con- 
vex. The  thin  place  on  the  cleft  side,  where  the  two 
thickened  ridges  jut  together,  facilitates  the  flattening  of 
the  cells,  while  they  curve  out  on  this  side.  Therefore, 
lest  the  movement  of  the  guard-cells  be  interfered  with, 
it  is  joined  to  the  epidermal  wall  on  a  suddenly,  narrowed 
side  and  is  fastened  to  it  after  the  manner  of  a  hinge. 
Under  the  stoma  is  the  breathing  cavity,  «,  of  the  same 
nature  as  the  larger  intercelhdar  spaces  filled  with  air, 
which  is  bordered  with  cells  containing  chlorophyll  and  is 
connected  with  the  open  cavities  found  between  these 
cells.  Testing  with  chloriodide  of  zinc  shows  us  that  the 
walls  of  the  epidermal  cells  are  colored  a  yellow-brown, 
through  their  whole  circumference,  with  the  exception  of 
a  thin,  somewhat  wrinkled  membrane,  and  are  the  so- 
called  cuticle,  c;  this  cuticle  swells  outward  at  the  stoma, 
forming  the  above  mentioned  beak-like  projection,  which 
stains  yellow-brown  ^v\Üx  the  chloriodide  of  zinc,  and  so 
appears  to  be  cuticularized.  As  an  extremely  delicate 
membrane,  the  cuticle  extends  through  the  stoma  over 
the  guard-cells  quite  to  the  beginning  of  the  parenchyma 
cells  containing  the  chlorophyll.  The  guard-cells  are  vio- 
let in  their  whole  extent.  By  the  application  of  concen- 
trated sulphuric  acid,  the  whole  section  is  dissolved  except 


64 


GUARD-CELLS    OF    STOMATA. 


the  cuticle  including  the  cuticularized  projection  of  the 
stoma. 

An  unusually  favorable  ol)ject  for  studying  the  stomata 
apparatus  is  found  in  the  Tradescanlia  virginica.  The 
epidermis  is  formed  of  polygonal  cells  mostly  somewhat 
extended  in  the  direction  of  the  leaf.  With  these  alternate 
narrow  stripes  of  slenderer  and  longer  cells.  These  stripes 
appear  green  on  the  under  side  of  the  leaf,  the  others  gray. 
The  lateral  walls  of  the  epidermal  cells  are  furnished  with 
pores,  the  outer  surface  is  faintly  striated.     The  number  of 


Fig.  27.    Epidermis  of  the  imderslcle  of  the  leaf  of  Tradescanlia  vircjinica.    A, 
surface  view;  JS,  transection;  I,  leucoplasts.    X  240. 


stomata  is  considerably  greater  on  the  under  than  on  the 
upper  side  of  the  leaf,  so  we  will  examine  that  side.  The 
stomata  are  almost  always  surrounded  by  four  cells.  Fig. 
27.  The  guard-cells  lie  at  the  same  height  as  the  epider- 
mis. The  cleft  which  lies  between  is  relatively  large. 
They  have  chlorophyll  grains  between  which,  for  the  most 
part,  the  nucleus  is  visible  ;  also  in  the  epidermal  cells  the 
nucleus  is  sharply  distinct,  surrounded  with  colorless  leu- 
coplasts, I,  Fig.  27,  A.  The  cell-sap  of  the  epidermal 
cells  is  here  and  there  rose-colored.     Make  a  cross  section, 


EPIDERMIS.  65 

as  with  the  last  example,  and  we  shall  have  the  stoma  as 
in  Fig.  27,  B. 

The  cleft  side  of  the  cells  has  thicker  walls  :  the  other, 
thinner.  The  walls  of  the  next  adjoining  cells  are  thinner 
than  the  succeeding  cells  of  the  epidermis,  and  are  the  sec- 
ondary cells  of  the  stoniata  apparatus,  forming  the  hinge, 
otherwise  provided  for  in  the  Iris,  as  we  have  seen.  The 
leucoplasts,  I,  surrounding  the  nucleus  are  very  favorably 
situated  for  examination.  It  is  interesting  to  note  that 
though  the  leucoplasts  are  here  exposed  to  the  action  of 
the  strongest  light  they  remain  small  and  colorless  and  do 
not  change  to  chlorophyll  grains.  Tvadescantia  zebrina 
has  a  similarly  constructed  stomata  apparatus,  found  on 
the  under  side  alone.  The  cross  section  is  very  instructive 
even  if  not  very  easily  made  thin  enough.  The  epidermal 
cells  of  both  sides  are  of  considerable  size,  particularly 
those  of  the  upper  side,  which  are  so  high  that  they  alone 
make  nearly  half  the  thickness  of  the  leaf.  Many  of  them 
are  divided  by  transverse  walls.  Mostly  the  epidermal 
cells  have  only  watery  cell-sap,  which  on  the  underside  of 
the  leaf  is  colored  red.  The  epidermal  cells  of  this  plant 
constitute  a  water-holder  of  gieat  capacity.  The  four  sec- 
ondary'^ cells  of  the  stomata  are  quite  flat  so  that  a  larire 
breathing  cavity  of  the  height  of  the  epidermal  cells  exists 
under  the  stomata.  Taking  a  section  from  the  under  sur- 
face of  the  leaf,  w^e  can  focus  down  throuo-h  it  and  sfet  a 
good  image  of  it  while  no  air -gets  into  it.  The  leuco- 
plasts about  the  nucleus  are  distinctly  seen  in  the  epider- 
mal cells. 

The  Aloe  and  Agave  species  show  an  exti-aordinary  de- 
velopment of  the  outer  wall  of  the  epidermal  cells  and  a 
corresponding  depression  of  the  stomata  deep  in  the  epi- 
dermis. 

Take  the  Aloe  nigricans  found  in  greenhouses ;  other 

5 


66 


STOMATA. 


species  of  the  genus  may  be  used  in  lack  of  this.  A  su- 
perficial section  shows  the  epidermal  cells  of  both  sides 
of  the  leaf  to  be  regularly  polygonal — mostly  hexagonal. 
The  cell  cavity  is  a  relatively  small,  oval  space;  it  is  us- 
ually filled  with  air  and  is  black  in  the  section.  The  sto- 
mata  occur  on  both  sides  of  the  leaf  at  the  bottom  of  the 
deep  clefts  which  are  surrounded  by  four  cells  and  are 
rectangular  and  enclosed  in  a  somewhat  projecting  rim. 
In  order  to  see  the  guard-cells,  the  section  must  belaid  on 
the  slide  inside  up ;  these  cells  are  relatively  broad  and 


Fig.  28.    Transection  through  the  epidermis  and  stoma  of  Aloe  nigricans,    i,  in- 
ner  thickening  layer.    X   240. 


short  and  contain,  among  other  things,  strongly  refractive, 
globular  oil-drops.  The  epidermis  being  so  hard,  we  may 
use  cork  in  making  the  section  ;  make  the  section  not 
the  whole  thickness  of  the  leaf,  but  from  one  side  only, 
cutting  from  the  soft,  inner  part  of  the  leaf  toward  the 
outer  and  harder,  making  the  section  perpendicular  to  the 
axis  of  the  leaf.  The  very  great  thickening  of  the  cells 
of  the  epidermis  is  at  once  seen  in  this  section,  Fig.  28. 
The  thickening  appertains  exclusively  to  the  outer  wall  of 
the  cell.     The  cell  cavity  runs  out  to  a  point  in  the  same 


STOMATA.  67 

direction  ;  this  thickened  wall  is  white,  strongly  refrac- 
tive "and  is  overspread  with  a  cuticular  membrane,  still 
more  refractive,  but  not  sharply  distinct.  The  lateral 
boundaries  of  the  cells  are  marked  only  by  a  delicate  line 
indicated  on  the  outside  by  a  slight  elevation  ;  the  inside 
of  this  thick  wall  is  covered  by  a  relatively  thinner,  less 
refractive  layer,  ^,  which  surrounds  the  cone-shaped  ex- 
tension of  the  cell  cavity,  and,  thinning  out  wedge-like, 
ceases  at  the  same  time  with  the  thickened  layer  on  the 
lateral  wall.  The  thickened  part  of  the  epidermis  in  this 
section  looks  like  a  notched  curtain.  Of  the  cavity  which 
leads  down  to  the  stoma  we  note  first,  the  projection  or 
rim  which  encloses  it,  and  also  that  the  tooth  which  forms 
the  thickened  layer  is  here  divided  on  one  side  and  loses 
half  its  height.  The  guard-cells  have  a  ridge-like,  and, 
in  section,  beak-shaped  projection,  both  above  and  on  the 
cleft  side ;  above  the  guard-cells  is  a  thin  place  in  the 
wall  which  forms  a  membranous  hinge  or  joint.  The 
breathing  cavity  is  deep  and  narrow. 

Parallel  striation,  more  or  less  oblique,  may  often  be 
seen  on  the  thickened  wall ;  it  is  caused  by  the  knife  in 
cutting  and  often  recurs  in  hard,  elastic  substances  in  the 
same  manner.  With  chloriodide  of  zinc,  the  thick  wall 
is  colored  yellow-brown,  showing  it  to  be  cuticularized. 
The  inner  laj'er,  ^,  and  the  rest  of  the  leaf  tissue,  are  col- 
ored violet.  The  yellow-brown  color  extends  through 
the  hinge  to  the  two  projections  on  the  guard-cells  ;  the 
remaining  part  of  these  cells  is  tinged  violet.  Sulphuric 
acid  dissolves  all  that  the  last  reao;ent  has  not  colored 
yellow-brown,  and  this  it  dissolves  in  an  hour,  leaving 
only  the  delicate  cuticle  and  the  fine,  middle  lamella  oc- 
curring between  the  epidermal  cells.  The  cuticle  extends 
over  the  guard-cells  to  the  point  where  they  join  the  inner 
cells  containing  chlorophyll.     The  cuticle  and  the  epider- 


68 


STOMATA. 


mal  layer  are  colored  brown  by  the  acid.  The  oil  in  the 
guard-cells  gathers  together  in  a  strongly  refractive  ball 
on  the  entrance  of  the  acid  and  after  some  time  disap- 
pears. 

In  the  arrangement  of  the  stomata  within  the  epider- 
mis many  modifications  occur.  A  very  remarkable  one 
is  that  when  the  stomata  are  surronnded  by  a  single  ring- 
shaped,  epidermal  cell.  The  fern  Aneimia  fraxinifolia^ 
found  in  every  botanic  garden,  will  show  this.  The  cells 
of  the  epidermis  have  an  extremely  wavy  outline,  Fig. 
29,  and  gain  stability  b}^  this  dovetailing  of  the  edges, 
so  common  in  epidermal  cells. 

In  the  ferns,  the  epidermal  cells  are 
richly  furnished  with  chlorophyll  grains. 
The  epidermis,  therefore,  belongs  with 
the  organs  of  assimilation  as  it  does  not 
in  most  phanerogams.  The  stoma  is 
set  in  the  surrounding  epidermal  cells 
as  in  a  rim.  A  transverse  section  shows 
it  to  be  raised  somewhat  above  the  sur- 
face of  the  epidermis  ;  this  extreme  case 
is  connected  by  transitional  forms  with 
others,  not  treated  here,  much  less  re- 
markable. We  should  accustom  our- 
selves to  think  of  the  stoma,  as,  in  fact, 
<)nly  inserted  on  the  side  walls  of  the  surrounding  epi- 
dermal cells;  then  the  singularity  of  its  insertion  will 
cease. 

Nerium  oleander  shows  a  peculiar  form.  Stomata  will 
not,  at  first,  be  found  either  on  the  upper  or  under  side, 
but  on  both  sides  a  uniformly  small-celled  epidermis, 
which,  particularly  on  the  under  side,  is  covered  with 
single-celled  hairs,  the  walls  so  thickened  as  alfliost  to 
obliterate  the  cell  cavity.      On  the  under  side  of  the  leaf 


Fig.  29.  Aneimia 
fraximfolla.  Stoma 
suiToumlert  by  epider- 
mal cells,  n,  nucleus 
of  epidermal  cells.  X 
210. 


WATER   STOMA. 


69 


are  found  certain  cavities  filled  with  air,  and  having  on 
their  edges  these  before-mentioned  short  hairs  ;  the  hairs 
interlock  across  the  opening  and  so  close  up  the  cavity  in 
front.  A  second  superficial  section,  taken  from  the  same 
place  whence  the  epidermis  has  already  been  removed, 
will  enalde  us  to  get  a  look  here  and  there  into  the  depth 
of  these  cavities.  Perhaps  an  air-pump  will  be  necessary 
to  remove  the  air,  or  else  an  immersion  of  the  section  in 
alcohol  ;  there  will  be  seen  projecting  from  the  walls  of 
this  cavity  small,  con- 
ical elevations,  the  \^ — <  °X^A===_/(^y 
apex  of  which  will  be 
formed  of  a  stoma. 
The  lateral  walls  of 
these  small  cones  con- 
sist of  epidermal  cells 
which  have  a  breath- 
ing cavity  between 
them  extending  to  the 
stoma.  The  same  kind 
of  hairs  which  we  saw 
on  the  edge  of  this  cav- 
ity spring  from  its 
walls  between  the 
cones. 

We  will  now  glance  at  the  water  pore  or  water  stoma. 
It  exhibits  a  structure  like  that  of  the  air  stoma,  on]y  it 
is  larger,  the  cleft,  together  with  the  intercellular  space, 
at  times  at  least,  being  filled  with  water  ;  the  guard-cells 
of  this  water  pore  may  be  unmovable  from  the  beginning, 
quickly  die  and  then  in  all  cases  lose  their  movability. 
The  best  object  for  studying  these  structures  is  the  Tro- 
pceolitm  maj'us.  The  water  pore  is  found  on  the  u[)per 
side  of  the  leaf,  and,  indeed,  on  the  ends  of  the  i3rincipal 


Fig.  30.  Water  stoma  and  surronmling  epi- 
dermal cells  from  the  edge  of  leaf  of  Tropieolum 
majus.    X    240. 


70  WATER    STOMATA. 

nerves  ;  here,  the  edge  of  the  leaf  commonly  exhibits  a 
small  depression.  One  may  approximately  see  the  water 
pore  by  putting  a  piece  of  the  leaf,  full  thickness  in  water, 
under  a  cover-glass,  and  place  it  under  the  microscope  ; 
but  the  peculiarities  of  it  will  really  be  recognized  only 
when  a  surface  section  be  made  of  this  part  of  the  leaf; 
it  will  then  be  seen  as  represented  in  Fig.  30.  The  con- 
tents of  the  guard-cells  are  reduced  to  a  minimum.  Sev- 
eral water  pores  are  always  found  but  a  short  distance  apart. 

NOTKS. 

(1)  Strasburger,  Jahrb.  f;  wiss.  Bot.  v,  p.  2Ö7;  de  Bary,  Vergl. 
Anat.,  p.  82  u.  ff.,  70  u.  fl'. ;  Schwendener,  Monatsber.  d.  Kgl.  Akad.  d. 
Wiss.  in  Berlin,  1881,  p.  833.  In  the  first  named  authors  will  be  found 
the  remaining  literature,  at  the  places  quoted. 

(2)  Westermaier,  Jahrb.  f.  wiss.  Bot,i  Bd.  Xiv^  p.  43. 


LESSON  VII. 
Epidermis.     Hairs.     Wax  and  Mucilage. 

We  are  already  acquainted  with  the  root-hairs  of  Hy- 
drocharis  morsus  ranee,  and  since  all  root-hairs  are  so 
much  alike  we  can  omit  further  consideration  of  them. 
We  have  also  seen  the 
conical  papill;«  or  elon- 
gated epidermal  cells  of 
various  petals.  So  also 
the  cask-like  cells  form- 
ing the  filamentous  hairs 
on  the  stamens  of  Tra- 
descantia,  Fig.  15  ;  also, 
finally,  the  hairs  of  the 
Cucurbita  with  its  many 
celled  base  running  out 
into  a  pointed  filament. 

The  manifold  aspect 
of  plant  hairs  is  already 
well  known  to  us,  but 
our  knowledge  of  them 
should  be  more  com- 
plete. We  meet  vari- 
ous forms  of  the  single- 
celled,  many  branched  fig.  31.  ^  and  5,  hairs  from  under  side  of 
,      ,  Ji        1  T     '^"'^  **'  Ckeiranthus  cheiri;  A,  hair  f?een  from 

naU'S  on  the  leaves  and  above.X  i'O-^.  transection.  X  210.  C,  hair  from 
stems  of   the    CrilciferrP      "'"'^r  side  of  leaf  of  3/«<</n'wZa  ajiwM«,  x  ^O- 

On  the  stem  and  leaves  of  the  common  wall-flower,  Chei- 
ranthus  cheiri,  Fig.  ?>l,  A,  we  have  a  lance-like  form  with 
the  cell  cavity  narrow,  and  disappearing  towards  the  end, 

(71) 


72  PLANT    HAIRS. 

the  surface  thicklv  beset  with  knobs  little  and  larsje.  The 
hair  lies  parallel  to  the  axis  of  the  plant,  and  so  a  good 
transverse  section  may  easily  be  made.  But  as  we  wish 
to  cut  it  through  the  middle,  at  the  point  of  its  insertion 
on  the  leaf,  a  number  of  sections  should  be  made  in  order 
to  get  just  the  one  we  want.  Thus  we  see.  Fig.  31,  B, 
that  the  place  of  insertion  lies  somewhat  deep,  and  that  the 
epidermal  cell  which  extends  outwardly  to  make  the  body 
of  the  hair  is  slenderer  than  its  neighbors,  swollen  out  and 
rounded  at  the  bottom  and  reaches  deeper  into  the  adjacent 
tissue,  forming  the  "foot"  of  the  hair.  A  longitudinal 
section  shows  that  the  foot  is  no  wider  in  the  other  dimen- 
sions than  in  this,  and  one  sees  clearly  that  the  cell  cavity 
of  the  foot  extends  without  interruption  into  the  body  of 
the  hair.  B}^  a  superficial  section  made  through  the  foot, 
we  shall  find  it  to  be  circular.  We  also  see  that  the  cells 
containing  chlorophyll  are  radially  arranged  al)out  and 
joined  to  that  part  of  the  foot  which  is  widened  and  ex- 
tended below  the  epidermis. 

Cheiranlhus  alpinus,  not  seldom  cultivated  in  botanic 
gardens,  presents  the  same  appearance  onl}'  that  at  one  or 
both  ends,  the  hair  forks  so  that  we  see  three  or  four  proc- 
esses all  spread  out  parallel  to  the  surface  of  the  leaf. 

The  hairs  of  the  stem  and  leaf  of  MattJiiola  annua  are 
repeatedly  branched  in  one  plane,  Fig.  31,  C.  These 
hairs  are  so  thick  upon  the  under  side  of  the  leaf  that  their 
branches  interlock.  The  walls  are  so  much  thickened  that 
the  cell  cavity  is  almost  obliterated.  Knobs  are  scarcely 
developed  on  the  surface.  The  ball-shaped  foot  of  the  hair 
is  considerably  swollen  and  the  cells  containing  chlorophyll 
are  beautifully  grouped  about  it  in  a  radial  manner.  A 
superficial  section  turned  over  will  show  this. 

The  long  single-celled  hairs  in  the  groove  of  the  spur- 
like prolongation  of  the  corolla  of  Viola  tricolor  have  a 


PLANT   HAIRS. 


73 


veiy  peculiar  form,  Fig.  32.  Make  a  cross  section  through 
the  lower  petal,  close  under  the  place  where  it  folds  up 
into  a  groove.  The  epidermal  cells  grow  out  into  a  hair 
for  almost  their  entire  lireadth.  The  hairs  are  covered 
Avith  irregular  knotty  swellings,  and  the  cuticle  with  lon- 
gitudinal projecting  ridges.  The  cell-sap  is  colorless,  but 
yellow  color-bodies  often  occur  in  the  wall-plasma. 

The  filament  of  the  stamens  of 
Verhascum  nigrum  is  covered  with 
single-celled  violet  hairs.  Remove 
the  anther  and  immerse  the  filament 
in  a  drop  of  water  on  the  slide.  The 
hair  is  quite  long,  swollen,  club- 
shaped  at  the  end  and  filled  with  a 
violet-colored  cell-sap.  It  is  covered 
with  longish  knobs  arransfcd  some- 
what  spirally  about  it. 

Branched,  many-celled  hairs  are 
found  on  the  under  side  of  the  edse 
of  the  corolla.  From  above  they 
have  a  certain  resemblance  to  those 
of  the  Matthiola,  but  in  this  case 
eveiy  branch  comes  out  of  a  common 
central  point,  and  consists  of  an  inde- 
pendent closed  cell.  It  also  branches 
not  in  one  plane,  but  at  all  angles. 
Its  cell  walls  are  as  thick,  but  there 
are  no  outside  projections  as  in  the  Matthiola.  On  the 
edge  of  the  leaf  the  hair  is  seen  in  a  lateral  view.  The 
body  of  the  hair  is  separated  fi-om  the  epidermal  cell, 
which  bears  it,  by  a  partition  wall.  It  consists  of  an 
almost  always  single-celled  stalk  and  the  attached  branches. 
The  edge  of  the  corolla  also  bears  glandular  hairs.  These 
consist  of  a  two-  or  three-celled  stalk  and  a  flattened  head, 


Fjg.  33.  Hair  from  the 
groove  in  tlie  petal  of  Viola 
tricolor.    X  210. 


74 


HAIRS    AND    SCALES. 


which  is  occasionally  covered  with  a  highly  refractive  sub- 
stance on  its  top.  The  latter  will  be  studied  elsewhere 
under  more  favorable  conditions. 

One  needs  only  to  think  of  the  branched  hairs  of  Ver- 
bascuni  nigrum  being  several  times  set  upon  each  other  in 
order  to  have  the  hairs  which  form  the  felt  on  the  leaves  of 
Verbascum  tJiapsiforme.  The  hairs  are  five  stories  high, 
each  story  being  separated  from  the  preceding  one  b}'  a 
single-celled  internode  which  is  a  continuation  of  the  prin- 
cipal axis  of  the  hair.     The  cells  are  mostly  filled  with  air. 


Fig.  33.    Scale  from  the  uncler  side  of  the  leaf  of  Shepherdia  canadensis.  A,  sur- 
face view;  /?,  sectional  view.    X  240. 

The  best  cross-section  is  made  through  the  mid-rib  of  the 
leaf. 

The  scales  of  Shepherdia  canadensis  belong  to  the  same 
category  as  the  branching  hairs  of  Verbascum.  With  the 
magnifying  glass,  we  shall  find  white,  loosely  built  and 
more  closelv  built  stars  on  the  under  side  of  the  leaf,  Fis^. 
33,  A.  On  the  upper  side  of  the  leaf  but  a  few  white 
stars  are  seen ;  the  cells  of  these  contain  only  air,  spring 
from  a  common  central  point,  but  are  laterally  separated 


THORNS   AND   SPINES.  75 

from  each  other.  On  the  upper  side  of  the  leaf  the  rays 
of  the  star  are  not  confined  to  one  plane,  but  radiate  in 
all  directions.  The  cells  or  rays  of  the  brown  stars  are 
connected  with  each  other,  almost  to  the  edge,  and  fur- 
nished with  living  contents.  The  nucleus  is  always  easily 
seen  within.  A  transverse  section  through  the  leaf,  where 
it  rightly  hits  the  brown  star,  shows  that  the  stalk  is  many 
celled.  Fig.  33,  B,  and  that  not  the  epidermis  alone,  but 
also  the  next  succeeding  laj^er  of  cells,  passes  into  it. 
The  stalk  bears  at  the  top  the  stellate,  single  lamellate, 
many-celled  expansion. 

In  lack  of  Shepherdia  canadensis,  Eleagniis  angustifolia 
may,  to  a  certain  extent,  be  substituted.  Here,  only  the 
white  scales  are  found  on  the  under  side  of  the  leaf,  the 
disk  consisting  of  cells  either  laterally  isolated  or  grown 
together  nearly  to  the  edge. 

Now,  make  a  lono^itudinal  cut  throuoli  the  stem  of  a 
rose,  perhaps  Rosa  semperßorens  of  the  garden,  at  a  place 
whence  a  thorij  projects  ;  if  possible,  make  it  so  as  to  di- 
vide the  thorn  in  halves,  and  then  make  the  thinnest  pos- 
sible section  ;  this  is  not  so  easy  to  do  ;  but  do  not  neglect 
in  cutting  to  moisten  the  surface  of  the  object. 

Having  made  a  section,  one  can  see  that  the  epidermis 
of  the  stem  continues  into  the  thorn.  The  cells  are  more 
thickened  and  elongated.  Beneath  the  epidermis  are  nar- 
row, greatly  thickened  cells,  and,  beyond  these,  cells  with 
wide  cell  cavities,  the  latter  filling  the  whole  middle  por- 
tion of  the  spine  ;  all  these  cell  walls  are  finely  perforated. 
The  epidermis  of  the  stem  is  separated  from  the  chloro- 
phyll tissue  beneath  b\'  a  layer  of  cells  without  chlorophyll, 
somewhat  thick,  elongated  and  joining  each  other  with 
sloping  sides.  These  cells  are  of  like  origin  with  those 
which  form  the  inner  tissue  of  the  spine ;  but  the  tissue 
elements  of  the  spine  are  separated  from  the  chlorophyll 
tissue  of  the  stem  by  a  flat-celled  tissue  layer;  this  tissue 


76 


STING   OF    NETTLE. 


layer  arises  by  division  from  the  lowermost  layer  of 
the  spine  tissue ;  it  follows  the  chlorophyll  tissue  of  the 
stem  but  a  little  way  and  then  turns  towards  the  epider- 
mis, so  as  to  work  off  the  lateral  edges 
of  the  base  of  the  spine  from  the 
chlorophyll-lacking  tissue  of  the  stem. 
It  is  the  one  cork  la3'er  nearest  to 
whose  outer  surfaces,  b}'  means  of  a 
separating  layer,  there  ensues  in  older 
parts  of  the  stem  the  separation  of  the 
spine  ;  so  the  spine  splits  away  from  the 
stem,  quite  smoothly  ah)ng  the  inner 
surface  of  the  cork  layer.  Spines  on 
the  petioles  lack  this  cork  layer. 

In  examininof  the  bark  tissue  ad- 
joining  the  spine  of  the  rose,  we  shall 
find  crystals  in  the  cells,  crystals  of 
calcium  oxalate  which  will  not  dissolve 
with  acetic  acid  or  potash,  but  with 
hydrochloric  acid  without  generating 
gas.  They  have  the  form  either  of  a 
monoclinic  column  or  of  a  drusen  ;  the 
latter  consists  of  a  great  number  of 
crystals  accumulated  upon  an  original 
crystal;  these  crystals  surprise  us  l)y 
their  size  and  their  stelhite  form. 
J'°.el,'^«2fSc:;  I"  order  to  obtain,  u„harn,ed,  tl,e 
also  epidermis  and  small  stinging  hairs  of  the  dioecious   nettle, 

hair.    X   90.  j^^  ^.      "-..    .  i.  i.    i       ^i  ,• 

Urtica  dioica,  we  must  take  them  trom 
the  3'ounger  parts  of  the  plant.  With  a  razor  cut  a  hair 
from  the  rib  of  a  young,  strong  leaf  and  put  it  in  water  on 
the  slide  ;  if  the  hair  is  dead,  it  will  be  found  to  be  filled 
with  air,  and  the  point  is  no  longer  intact.  The  unin- 
jured hair  is  represented  in  Fig.  34 ;  the  hair  is  single- 
celled,  sharply  pointed  and  the  point  swollen  to  a  small 


GLANDULAE    HAIRS.  77 

head.  At  the  base,  the  hair  is  broadened  like  a  retort, 
and  this  bulb  is  embedded  in  a  cup  formed  from  the  tissue 
of  the  leaf.  The  history  of  its  development  shows  the 
hair  to  be  derived  from  a  single  epidermal  cell  which  lay 
at  the  same  elevation  as  the  neighboring  cell,  and  the 
greatly  swollen  foot  of  the  hair  was  lifted  up  upon  a  col- 
umn covered  with  epidermal  and  formed  of  snb-epidermal 
tissue.  In  the  hair  itself,  protoplasmic  streaming  may  be 
observed.  The  nucleus  is  commonly  found  suspended  in 
the  bulb  by  plasma  threads.  The  cuticle  is  covered  with 
oblique  ridges.  The  walls  of  the  hair  are  silicated  as  may 
be  shown  by  incinerating  it  on  a  mica  plate.  One  often 
finds  hairs  with  the  points  broken  off.  By  carelessly 
touching  the  hair,  its  point  is  driven  into  the  skin,  and, 
as  it  is  very  brittle,  it  breaks  off,  and  the  strongly  acid 
sap  flows  into  the  Avound  and  causes  a  slight  inflammation. 
A  small,  single-celled,  bristle-like  hair  is  seen  near  the 
other,  Fig.  34,  distinguished  by  its  fine  point  and  its  thick 
walls  ;  these  bristles  may  be  seen  on  the  edges  of  the  leaf. 
Put  a  bit  of  the  leaf  under  the  cover-glass  in  a  drop  of 
water.  In  all  leaves  the  bristles  will  be  seen  from  which 
the  cell  cavity  has  almost  disappeared;  their  surfaces  are 
covered  with  small  knobs. 

Glandular  hairs,  mentioned  in  connection  with  Verbas- 
cum  nigrum,  may  be  studied  under  most  favorable  con- 
ditions in  Primula  sinensis  —  primrose.  jNIake  a  tran- 
section of  a  petiole.  The  body  of  the  hair  is  separated 
from  the  epidermoidal  foot-cells  by  a  transverse  wall 
outside  of  the  epidermis  and  forms  a  cell  fibre,  which  con- 
sists of  mostly  two,  sometimes  more,  long  and  ^vide  cells 
and  one  (rarely  two)  slenderer  and  shorter  cells;  these 
last  cells  bear  the  little  globular  head ;  but  upon  this  is  a 
cap  of  strongly  refractive,  resinous,  yellowish  substance. 
The  secretion  takes  place  between  the  cuticle  and  the  cell 


78 


GLANDULAR   TUFTS. 


membrane.  The  cuticle  is  raised  up,  distended  and  finally 
ruptured,  and  the  secretion  is  poured  out  over  the  top  of 
the  hair.  An  application  of  alcohol  removes  the  secretion, 
and  the  raised  cuticle  will  be  seen  lying  in  folds.  The 
cells  of  the  hair  show  a  beautiful  network  of  protoplasm 
with  suspended  nucleus  in  which  lie  large  nucleoli.  In  the 
wall  plasma  are  no  chlorophyll  grains. 

The  glandular  tufts  on  the 
membrane-like  prolongations 
of  the  leaf  sheaths  of  Riiraex 
jpati&ntia  are  extremely  inter- 
esting. The  matter  secreted 
by  these  tufts  is  so  consider- 
able that  in  moist  weather  the 
young  leaves  and  the  ends  of 
the  stems  are  covered  with 
mucilage.  To  examine  this 
membranous  sheath  direct, 
the  inside  should  be  turned 
upward.  The  tufts  will  look 
like  small  leaves,  Fig.  35. 
These  minute  leaves  originate 
with  a  short  single-celled  foot 
from  a  small  epidermal  cell. 
On  the  one  cell  are  two,  on 

epidermal  prolongation  on  the  i^Mmea;     tlieSC  mostly  four,  wllich    CX- 

po  itntta.  X  240.  tending  in  the  direction  of  the 

Jong  axis  of  the  leaflet  are  repeated  several  times.  On 
the  further  side-walls  of  the  tuft  are  often  seen  bladder- 
like protrusions  which  occupy  a  part  or  the  whole  of  the 
wall  of  a  cell.  The  mucilage  is  formed  here  also  I)etweeii 
the  cuticle  and  the  rest  of  the  cell  membrane,  and  lifts  the 
cuticle  up.  The  bladder  finally  opens  and  lets  the  muci- 
lage out.     This  is  not  colored  with  iodine  or  chloriodide 


DIGESTIVE    GLANDS. 


79 


of  zinc.  In  water  it  swells  to  a  perfectly  clear  solution 
and  behaves  itself  like  a  gummy  body.  The  cells  of  the 
tuft  are  rich  in  protoplasmic  contents  and  their  nucleus 
is  very  distinct.  With  rose  aniline  violet  the  tuft  is  stained 
an  intense  violet,  the  mucilage  mass  becomes  a  pale  red. 
Aqueous  nigrosin  solution  stains  the  mucilage  steel  blue 
without  colorino-  the  tuft. 

The  structure  of  the  glandular  hairs  hav- 
ino^  the  function  of  tentacles  and  digestive 
organs  of  the  Drosera  rotundifoUa  is  also 
very  interesting.  They  arise  from  the 
edges  and  the  whole  upper  surface  of  the 
leaf  in  the  form  of  slender  filaments  attenu- 
ated upwards  and  expanded  into  an  egg- 
shaped  termination,  Fig.  36.  They  are 
constructed  of  delicate  elongated  cells,  the 
larger  hairs  penetrated  throughout  with 
one  or  more  spirally  thickened  tubes,  the 
spiral  vessels.  The  radial  extension  of  the 
epidermis  to  form  the  head  of  the  hair, 
the  superficial  arrangement  of  the  epidermal 
elements  and  their  increase  till  they  consist 
of  three  layers,  may  be  best  seen  in  an  op- 
tical section  of  the  object,  Fig.  36.  The 
number  of  the  spirally  thickened  cells  are 
greater  in  the  head  of  the  hair.  All  cells 
which  lie  inside  the  envelope,  formed  by 
the  division  of  the  epidermal  cells,  are  spi-giami  oi  Drosera  ro 
rally  thickened.  By  examination  of  the 
place  of  insertion,  we  shall  see  that  not  only  the  epider- 
mis but  also  the  inner  tissue  of  the  leaf  is  continued  into 
the  hair.  These  digestive  glands  secrete  a  sWmy  fluid 
which  clings  to  the  top  of  the  little  head  like  a  drop  of 
•dew.     It  is  not  produced  from  beneath  the  cuticle  but  on 


Fig. 


36.    Digestive 


80 


WAX-SECRETIXG   TUFTS. 


the  free  outer  surface.  Small  insects  get  caught  and  stuck 
fast  in  this  slimy  drop  and  then  by  the  bending  down  of  the 
hairs  they  are  carried  to  the  middle  of  the  leaf.  Now  other 
hairs  also  bend  over  and  touch  the  insect  body  with  their 
tops.  Immediately  the  chemical  qualities  of  the  secretion 
change,  a  free  acid  and  a  pepsin-like  ferment  being  pro- 
duced which  slowly  digests  the  insect  or  any  other  albu- 
minous I)ody.  The  digested  substance  is  then  absorbed 
into  the  plant. 

A  transection  throusjh  the  winter  bud  of  the  horse-chest- 
nut,  ^scidus  hij)pocastanum,  shows  us  button-shaped  glan- 
dular tufts  upon  the  covering 
scales,  Fig.  37.  The  middle 
scale  has  these  tufts  on  both 
sides,  but  the  outside  scales 
have  these  more  on  the  inner 
surface,  and  the  inside  scales 
more  on  the  outer.  The  struct- 
ure of  the  tuft  is  seen  from  the 

FIG.  37.    Glandular  tuft  from  an  en-    ßauve  ;    a  SericS  of  Cclls  in  the 

veloping  scale  of  the  winter  bud  of       ~      ^ 

yEscuius  hippocastmmm  surrounded  middle  divide  above  and  from 
by  its  secretion.  X  240.  ^j^^^^  ^^^^  sccretiiig  ccUs  radi- 

ate. The  illustration  shows  a  longitudinal  section  of  the 
tuft.  The  cuticle  will  burst  and  the  secretion  will  be 
poured  out  between  the  scales  coating  them  over  andglu- 
ins:  them  together.  This  secretion  is  a  mixture  of  gum 
and  resin.  The  minute  drops  of  gum  swell  in  water,  while 
rose  aniline  violet  colors  the  resin  masses  a  beautiful  blue. 
The  contents  of  the  tufts  become  red. 

We  have  already  remarked  the  fine-grained  waxy  coat- 
ing upon  the  epidermis  of  Iris  ßorentina.  We  wull  now 
investigate  this  point  in  some  other  plants. 

A  suitable  plant  is  found  in  Eoheveria  glohosa.  The 
w^axy  coating  gives  the  plant  a  hoaiy  or  glaucous  appear- 


WAX    INCRUSTATION. 


81 


ance,  and  may  be  easily  rubbed  ofl'  from  the  leaf.  An 
inspection  of  the  upper  surfiice  of  the  epidermis  shows  us 
a  net-like  crust  of  blended  grains. 

A  wax  coating,  in  the  form  of  short  rods  massed  together, 
may  be  easily  observed  on  the  epidermis  of  Eiwaliptus 
globulus. 

Saccharum  officinarum  is  also  a  beautiful  object.  Here 
the  wax  coating  appears  in  the  form  of  long  rods  often 
curled  at  the  ends.  Prepare  a  superficial  section  from  a 
node  of  the  stem  noticeable  for  its  glaucous  appearance.  To 
remove  the  air  from  between  the  rods  dip  the  section  in 


Fig.  3S.    Transection  through  a  node  of  the  stem  of  Saccharum  offlci-narum  show- 
ing a  rod-shaped  wax  coating.    X  510. 

cold  alcohol  for  a  short  time.  It  is  difficult  to  make  a 
transection  to  which  the  little  rods  still  adhere.  Such  an 
one  is  shown  in  Fig.  38.  The  rods  stand  closely  pressed 
together,  and  show  the  before  mentioned  curling.  Brought 
near  a  flame  the  rods  melt  as  they  also  do  in  hot  alcohol. 

Note. 


(1)     See  in  de  Bary's  Vergl.  Anat.  die  §§  10,  13,  16,  u.  ff.  aud  there 
also  for  the  literature. 


LESSON  VIII. 
Closed  Collateral  Vascular  Bundles. 

The  common  cornstalk,  Zea  Mays,  furnishes  an  excel- 
lent object  for  studying  the  structure  of  the  closed  collat- 
eral fibrovascuhir  bundles  of  the  monocotyledons  (1). 
An  alcohol  specimen  should  be  used  for  studying  the  cell 
contents.  Make  a  transection  through  an  internode  and 
put  it  on  a  slide  in  a  diop  of  chloriodide  of  zinc  and  lay 
the  slide  on  a  piece  of  white  paper.  We  may  now  with 
the  naked  eye  see  the  bundles,  in  the  form  of  oval-shaped 
dots,  and  their  arrangement  characteristic  of  the  mono- 
cotyledons. There  is  no  specialization  of  pith  and  rind 
by  the  distribution  of  the  bundles.  With  a  low  power, 
select  for  examination  a  bundle  lying  not  too  near  the 
surface  of  the  stem,  noting  which  way  the  latter  lies  so  as 
to  distinguish  the  inner  and  outer  edge  of  the  bundle. 
The  bundle  is  represented  in  Fig.  39.  The  sheath,  vg, 
consists  of  thickened  lignitied  parenchyma  cells,  surrounds 
the  bundle,  and  is  stained  red-brown.  The  intercellular 
passage,  I,  is  surrounded  with  narrow  thin-walled  cells  col- 
ored yellow  by  the  zinc  reagent.  The  ring,  a,  belongs  to 
a  ring  vessel,  for  the  most  part  ruptured  by  stretching. 
The  intercellular  passage  may  be  produced  by  either  of 
two  ways,  by  the  rupturing  of  the  cells  or  by  their  separa- 
tion from  each  other.  The  one  we  may  call  the  "lysignian," 
the  other  the  "schizoginian,"  method.  The  ring  vessels 
and  some  other  which  sometimes  project  into  this  passage 
are  the  first  elements  formed  in  the  fibro vascular  bundles 
when  the  plant  is  rapidly  growing  in  length.     Upon  the 

(82) 


FIBRO-VASCULAR   BUNDLES. 


83 


outer  edge  of  the  l)imdle  are  one  or  more  vessels  ;  one  in 
this  case,  sp.  This  vessel  has  spiral  walls  as  we  shall  be 
able  to  see  by  a  longitudinal  section.  Further  on  at  the 
right  and  left  are  two  wide,  open  spaces,  m,  m',  vessels  with 
pitted  walls.     A  ring  or  part  of  one  is  often  seen  project- 


FiG.  39.  Section  of  a  flbro-vascular  bundle  from  the  inner  part  of  a  corn  stalk, 
Zea  Mays.  «Joint  of  a  ring  vessel;  sp,  spiral  vessel;  to  and  »*',  pitted  vessels;  i;, 
sieve  tubes;  s,  conducting-cells;;»-,  compressed  protophloem  elements;  I,  intercel- 
lular passage;  rgr,  sheath.    X  ISO' 

irg  into  the  interior  of  these  vessels,  m'.  It  is  all  that  re- 
mains of  a  perforated  division  wall  once  existing  intact 
between  two  cells.  The  cells  lying  between  these  large 
vessels  are  reticulated.     The  walls  of  the  vessels,  espec- 


84  VASCULAR   BUNDLES 

ially  of  these,  are  stained  a  yellow-brown.  The  cells  be- 
tween the  vessels  are  stained  a  darker  yellow  than  those 
al)out  the  intercellular  passage. 

The  above  described  portion  of  the  bundle  is  called  the 
woody  or  xylem  vascular  part ;  this  does  not  impl}^  that 
these  cell  walls  are  very  much  thickened.  The  woody 
part  or  vessels  are  never  lacking  in  the  bundle,  hence  the 
term  xylem  is  morphologically  correct.  In  the  example 
just  studied,  we  have  touched  upon  the  woody  portions, 
viz.,  the  xylem,  the  protoxylem,  the  wood  parenchyma 
and  the  vessels  of  the  tibrovascular  bundles. 

For  the  other  or  secondaiy  element  of  the  bundle,  we 
select  the  term  bast  or  phloem.  Since  the  sieve-tubes 
never  fail  in  the  bast,  it  is  morphologically  more  reason- 
able to  call  the  bast  the  sieve  part  (2).  Vessels  and  sieve 
tubes  con8titute  the  tibrovascular  bundles,  and,  being  lat- 
erally united,  they  are  called  collateral  bundles.  By 
including  the  sheath  also,  we  may  call  the  whole  a  iibro- 
vascular  cord  (3). 

A  solution  of  the  chloriodide  of  zinc  colors  the  bast  in 
this  bundle  a  distinct  violet.  Wide  and  narrow  unligni- 
fied  cells  regularly  alternate  :  the  former  are  sieve  tubes, 
V  ;  the  latter  conducting-cells,  s.  We  shall  see  the  fine, 
sieve-like  punctures  when  the  section  is  made  at  or  near 
a  division  wall  (see  illustration).  On  the  outer  border 
of  this  group  are  a  number  of  thick-walled  cells,  j^r; 
they  were  the  first  produced,  but  their  activity  terminated 
in  the  sieve-tubes  and  conducting-cells  ;  they  are  the  be- 
ginnings of  the  bast,  the  protophloem  elements,  and  are 
stained  brownish.  The  cells  of  the  sheath  border  upon 
these,  the  inner  ones  having  Avide  cell  cavities,  but  the 
outer  sclerenchyma  cells  of  the  sheath  gradually  pass 
over  through  intermediate  forms  into  the  large-celled  pa- 
renchymatous, fundamental  tissue ;  the  cells  of  the  latter 


OF   MONOCOTYLEDONS.  85 

are  stained  yellow  with  an  occasional  violet  tinge.  Towards 
the  outer  surface  of  the  stem  the  l)undles  are  more  closely 
packed,  the  intercelhilar  passages  disappear,  the  elements 
are  reduced  one  by  one,  and  the  sheath  is  greatly  en- 
larged. Lateral  unions  between  large  and  small  bundles 
are  frequent  in  this  part  of  the  stem,  the  conjunctions 
takinof  phice  on  the  sides  at  the  points  where  the  large 
vessels  are. 

Enclosing  the  epidermis  of  the  stem  is  a  stout  ring  of 
tissue,  like  that  of  the  sheath,  and  gives  the  same  reac- 
tion with  chloriodide  of  zinc.  This  extreme  outer  layer 
is  called  the  hypoderma  and  is  unbroken  except  by  the 
stomata.  The  hypoderma  and  the  bundle  sheath  protect 
the  thin-walled  tissue  and  give  stability  to  the  stem  ;  com- 
prehensiveW,  they  are  the  "stereids"  and  form  the  mechan- 
ical-tissue system,  the  "stereöms"  (4).  Mechanic.il  prin- 
ciples require  that  for  stiffness  the  stereöms  be  [)laced  as 
near  as  possible  to  the  periphery  of  the  stem.  The  pe- 
ripheral bundles,  including  the  sheaths,  constitute  a  system 
of  compound  pillars.  The  sheath  is  the  column,  the  bun- 
dle is  its  feeling.  The  hypodermal  cylinder  is  strength- 
ened by  the  sclerenchyma  tissue  of  the  sheaths,  even  if,  as 
in  this  case,  it  is  not  much  developed  ;  this  cylinder  con- 
sists of  a  number  of  interblended  columns  placed  in  a 
circle. 

Coralline  soda  (5)  rapidly  stains  the  lignified  walls  in 
both  the  bundles  and  fundamental  tissue  a  bright,  coralline 
red,  and  the  unlignified  a  rose  color.  This  brings  into 
great  prominence  the  sheath  cells,  the  walls  of  the  vessels 
and  the  epidermal  ring. 

Now,  make  a  number  of  radial  sections,  so  as  to  be  sure 
to  have  one  at  least  through  the  middle  of  a  bundle.  It 
should  show  the  bast  and  the  ring  vessels  ;  stain  with  chlor- 
iodide of  zinc;  the  colors  will  correspond  to  those  of  the 


86 


VASCULAR    BUNDLES 


transection.  But  a  coralline-stained  section  will  be  bet- 
ter for  study,  Fig.  40.  Begin  at  the  inner  edge  of  the 
bundle.  We  first  meet  the  nearly  cubical  cells  of  the  fun- 
damental tissue,  and  then  the  sheath  cells  of  the  bundle, 
vg,  deeply-stained,  elongated,  somewhat  pointed,  and 
dotted   with  slit-like  pits  diagonally  arranged.     Within 


Fig.  40.  Longitudinal  section  of  corn  sttilk.  Zea  Mays,  a  and  «',  joints  of  a  ring 
vessel;  sp,  spiral  vessel;  v,  sieve  tubes;  s,conducting-cells;^?-,  protophloem ;  I,  air 
passage ;  vg,  sheath.    X  180. 

these  elongated  sclerenchyma  cells  is  a  much  reduced  wall 
layer  of  protoplasm  and  a  nucleus.  Next  to  the  sheath 
is  the  intercellular  passage  which  extends  through  the 
whole  length  of  the  bundle.  It  is  surrounded  Avith  pri- 
mary, wood-parenchyma  cells,  thin-walled,  shorter  than 
those  of  the  sheath,  have  more  cell  contents  and  are  sep- 
arated by  transverse  walls.     Isolated,  small  rings,  a,  be- 


OF   MONOCOTYLEDONS.  87 

lonofino;  to  a  rinoc-vessel  which  has  beeu  ruptured  durino; 
the  elongation  of  the  iuternode  are  seen  attached  to  the 
outer  side  of  the  passage  ;  these  and  other  small  rings, 
sometimes  seen,  a' ,  constitute  the  remainder  of  the  pro- 
toxylem  elements.  Next  to  the  rings  are  one  or  more 
spiral  vessels,  sj) ;  only  one,  and  that  a  narrow  one,  in  this 
case.  Next,  are  somewhat,  thicker- walled,  relatively 
short,  wood  parenchyma  cells,  with  pitted  and  mostl}''  re- 
ticulated walls  ;  beyond  these  are  the  bast  cells,  recog- 
nized by  their  thick,  rose-colored  division  walls,  the 
sieve-plates  of  the  sieve-tubes,  v,  the  punctures  of  which 
may  be  seen  with  a  high  magnifying  power.  The  con- 
ducting cells,  s,  are  placed  side  by  side  with  the  sieve-tubes, 
are  slenderer,  shorter,  and  have  a  nucleus  which  the  sieve- 
tubes  have  not.  The  sheath  cells  bound  the  vascidar  bun- 
dle and  are  so  acutely  pointed  that  we  may  speak  of  them 
as  vascular  fibres.  Starch  grains  are  not  found  in  the  cells 
of  the  vascular  bundles  nor  in  those  of  the  fundamental 
tissue.  All  these  cells  except  the  vessels  and  the  sieve- 
tubes  have  a  nucleus. 

A  longitudinal  section  through  the  middle  of  the  bundle 
will  not  show  the  larger  vessels,  except  in  some  cases  by 
deep  focussing,  and  then  indistinctly.  To  see  these,  make 
a  section  through  the  side  of  the  bundle.  They  are  ob- 
liquely pitted.  The  pits  are  enlarged  at  the  bottom  but 
bordered  only  on  one  side,  as  the  corresponding  pits  of 
the  adjacent  wood-parenchyma  cells  have  no  borders. 
The  diaphragm,  which  consists  of  a  double  ring  projecting 
into  the  interior  of  a  vessel,  is  produced  by  the  thickening 
of  the  outer  edge  of  the  transverse  wall  of  the  cell,  and 
the  subsequent  absorption  of  the  thin  middle  partition. 

We  should  now  make  a  permanent  preparation  of  both 
the  transverse  and  longitudinal  sections.  Stain  with  saf- 
Iranin  or  iodine  green.    For  double  staining,  immerse  for 


88 


VASCULAR   BUNDLES. 


a  considerable  time  in  iodine  green  and  then  in  Grenadier's 
carmine  (G) .  For  instantaneous  double  staining  use  picro- 


FlG.  41.  Transeotion  of  flbro-vascular  bundle  from  leaf  of  Iris  florentina.  Ele- 
ments with  daik  outlines  ar«  vessels  and  the  shaded  ones  are  those  rich  in  cell 
contents,  ss,  compressed  spiral  vessels;  sp,  wide  spiral  vessels;  sc,  scaliform  ves- 
sels ;  V,  sieve  tubes  and  between  them  the  narrow  conductiug-cells ;  pr,  compressed 
protophloem  elements;  vg,  sheath  with  wavy  radial  cell  walls;  k,  section  of  a  crys- 
tal.   X-W. 

nigrosin,  or  picro-aniline  blue.     The  iodine  green  and  the 
picric  acid  will  color  the  lignihed  cell  walls  ;  the  carmine, 


STAINING    SECTIONS.  89 

nigrosin  and  aniline  blue  stain  the  unlignified  walls  and 
the  cell-contents.  Mount  in  glycerine  or  glycerine  jelly. 
If  in  the  former,  clean  away  all  the  superfluous  glycer- 
ine and  cement  the  cover-glass  down  with  a  solution  of 
Canada  balsam  in  turpentine  or  chloroform,  of  the  consist- 
ency of  syrup.  Use  a  glass  rod  as  thick  as  a  match  for 
this  purpose.  Gold  size  or  varnish  will  not  answer.  Gly- 
cerine-jelly mounts  do  not  need  to  be  sealed.* 

If  a  cornstalk  is  not  available  for  this  examination  use 
the  stem  oi  Avena  saliva ,  common  oat,  or  any  other  Gram- 
inacece. 

We  will  next  make  a  longitudinal  and  transverse  section 
of  an  alcohol  specimen  of  the  full-grown  leaf  of  Iris  flor- 
eniina.  The  alcohol  specimen  cuts  better,  contains  no 
air  and  has  the  cell-contents  already  fixed.  Let  it  lie  for 
some  time  before  cutting  in  a  mixture  of  glycerine  and  a|- 
cohol.  Stain  the  section  by  immersion  for  several  hours 
in  borax-carmine,  and  a  short  time  in  iodine  green.  The 
cell- contents  will  take  the  carmine.  The  lio^nified  walls 
and  vessels  will  be  stained  green,  also  commonly  that  part 
of  the  sheath  which  lies  next  to  the  bast.  The  proto- 
phloem  elements  will  be  blue.  Such  a  section  is  represented 
in  Fig.  41.  The  cells  with  red  cell-contents  are  shaded 
in  the  ilhistration.  The  green  walls  of  the  vessels  are 
dark,  and  the  blue  protophloein  cells  are  light.  The  thick- 
ened cells  of  the  fundamental  tissue  are  unlignified  and 
hence  uncolored.  For  instantaneous  staining  use  only 
iodine  green,  and  if  only  the  lignified  walls  are  to  be 
stained  the  exact  time  for  the  efiect  must  be  carefully  de- 
termined. The  cell-contents  in  that  case  Avill  not  be 
stained. 

Our  examination  will  proceed  from  the  wood  towards  the 
bast  elements  from  the  inner  and  upper  to  the  outer  and 

*But  they  may  be  moi-e  conveuiently  handled,  and  are  less  liable  to  injury  if 
they  are  securely  cemented  and  nicely  finished.— A.  B.  H. 


90  VASCULAR   BUNDLES. 

lower  side  of  the  leaf.  The  number  of  vessels  is  pretty 
large  in  the  wood  part  and  they  increase  in  size  towards 
the  bast.  They  either  touch  each  other,  or  are  separated 
by  thin  walled,  relatively  narrow,  primary  wood-paren- 
chyma cells  full  of  cell-contents.  These  cells  also  sur- 
round the  vessels  and  separate  them  from  the  fundamental 
tissue.  The  compressed  cells,  ss,  are  protoxylem  elements. 
In  the  bast  the  wdde  cells  are  the  sieve-tubes,  and  the 
small  ones,  rich  in  contents,  are  the  conducting-cells.  Be- 
yond, at  pr,  lie  the  protophloem  elements,  stained  blue. 
This  part  of  the  bast  is  surrounded  with  the  much  thick- 
ened sclerenchyma  of  the  sheath.  It  is  wanting  about 
the  rest  of  the  vascular  bundle.  The  cells  of  the  funda- 
mental tissue  are  smaller  about  the  vascular  bundle,  and 
have  no  intervening  air  spaces.  There  are  several  inter- 
mediate forms  between  these  cells  and  the  larger  ones  of  the 
fundamental  tissue  with  air-filled  intercellular  spaces.  At 
Ä;  is  a  small  cell  containing  a  highly  refractive  crystal. 

By  the  use  of  coralline,  we  shall  get  a  good  and  rapid 
staining,  the  lignified  sclerenchyma  becoming  a  fiery  red, 
the  as  3^et  unlignified  a  bright  rose  color,  the  vessels  a 
brown  red  and  the  other  elements  a  pale  3'^ellow  red. 

For  comparison  we  will  prepare  a  section  from  a  fresh 
leaf.  We  see  that  the  outer  large  fundamental  cells  con- 
tain chlorophyll ;  those  next  the  bundle  do  not.  The  ves- 
sels contain  air  which  somewhat  impairs  their  microscopic 
image.  The  radial  walls  of  the  first  layer  of  cells  next 
the  wood  part  of  the  bimdle  seem  to  have  dark  broad  pits. 
By  looking  at  the  other  section  again  Ave  see  that  these 
walls  are  arched  towards  one  side,  vg,  Fig.  41.  By  focus- 
sing up  and  down  we  shall  see  that  this  arched  part  of  the 
wall  forms  a  wave-like  band  bent  back  and  forth.  As 
we  shall  meet  a  similar  structure  elsewhere  we  will  spend 
no  more  time  on  it  now. 

A  longitudinal    section  cut  through  the   middle    of  a 


CRYSTALS    IN    CELLS. 


91 


bundle  shows  on  the  inner  edge  of  the  bundle  a  much 
elongated,  partly  compressed,  spiral  vessel.  Fig.  41,  ss; 
this  is  the  protoxylun  element.  Beyond  are  closely 
wound  spiral  vessels,  and,  further  along,  narrow,  scale- 
formed  vessels.  In  the  bast  the  sieve-plates  show  plainly 
only  in  the  coralline  preparation.  Further  towards  the 
outside  are  the  sclerenchyma  fibres  distinguished  by  their 
consideral)le  length,  thickness  and  pointed  ends. 


V 


Jf 


Fig.  42.  ^.crystal  of  calcium  oxalate  in  acellof  theleaf  of /WsJ^oj-en^jn«.  X240. 
B-B,  figures  which  explain  the  form  of  the  crystal;  B^  and  B\  and  D,  the  optical 
section  ;  C,  projection  on  a  symmetrical  plane. 

In  the  longitudinal  section,  the  crystals  are  seen  in 
profile,  lying  parallel  to  the  longer  axis  of  the  leaf.  Fig. 
42,  A-B;  they  are  found  in  elongated  cells  of  the  funda- 
mental tissue,  the  crystals  being  nearly  as  long  as  the 
cells ;  these  cells  have  no  chlorophyll  like  most  of  the 
neighboring  cells.  The  crystals  are  calcium  oxalate  and 
dissolve  without  generating  gas  in  muriatic  acid;  they 
have  a  long  prismatic  form,  are  mostly  twins,  D,  and  be- 


92 


CLOSED  VASCULAR    BUNDLES. 


ph~ 


-yiii 


long   to  the   monoclinic    system.       The    cell-contents    of 
these  cells  are  not  stained  with  the  coralline. 

The  vascular  bundles  of  the  monocotyledons  are  mostly 
built  on  the  type  of  these  two  cases.  Closed,  vascular  bun- 
dles are  not  well  adapt- 
ed to  the  lateral  orowth 
of  monocotyledons.  This 
growth,  which  is  limited 
to  plants  of  the  families 
of  Draccenece,  Ali  one  ce 
and  Dioscwacece,  takes 
place  by  means  of  a  cam- 
bium zone  lying  outside 
of  the  vascular  bundles. 

We  will  select  Dra- 
coena  rubra,  cultivated  in 
every  market  garden,  and 
make  a  transection  of  the 
same.  Inside  the  I)rown 
cork  layer,  we  see  with 
the  naked  eye  a  green,  soft 
rind  about  1  mm.  thick, 
ao-ainst  which  is  contrast- 
ed  the  yellowish  hard 
tissue  of  the  stem.  On 
the  border  of  this  is  the 
cambium  rino-.  The  cir- 
cular  centre  of  the  stem 
has  a  lighter  color. 

Now  put  the  section 
under  the  microscope,  wdth  a  low  magnification,  Fig.  43. 
The  central  fundamental  tissue  of  roundish  cells,  m,  has 
in  it  isolated  round  or  oval  vascular  bundles,  ß.  Beyond 
a  certain  point,/",  they  are  more  numerous,  compressed 


Fig.  43.  Transection  of  stem  of  Draccena 
rubra.  /,vascular  bundle;/', primary. .r',  sec- 
ondary bundles;/"',  leaf  bundles;  m,  imlig- 
nifled  fundamental  tissue;  s,  lignifled  funda- 
mental tissue,  surrounding  the  bundle  ;<,  tra- 
cheids;c,  cambium  ring;  cr,  rind;  I,  cork;pÄ, 
cork  cambium;  r,  bundles  of  raphides.  X^O. 


CLOSED  VASCULAR  BUNDLES.  93 

and  crowded  together  so  as  to  touch,  or  are  separated  by 
only  a  single  layer  of  fundamental  tissue.  In  the  latter, 
the  cells  are  much  thickened,  deeply  pitted,  radially  elon- 
gated, and  arranged  in  a  radial  series.  Beyond  this  we 
come  to  the  boundary  between  the  yellow  tissue  and  the 
green  rind,  c,  a  zone  of  flattened,  thin-walled  cells  ar- 
ranged in  a  strictly  radial  order.  This  is  the  cambium 
ring  which  provides  for  the  lateral  or  radial  growth  of  the 
stem.  It  belongs  apparently  to  the  fundamental  tissue. 
In  the  middle  of  the  zone  is  the  initial  layer  of  cells,  one 
cell  thick,  whose  successive  cell  divisions  produce  the 
new  elements.  These  divisions,  occurring  tangentially, 
produce  the  radial  arrangement  of  cells.  An  occasional 
radial  division  occurs  also,  and  so  starts  a  new  radial  series 
of  cells.  Vascular  bundles  occur  in  the  young  tissue  in  all 
stages  of  development.  The  youngest  consist  of  a  group 
ot  thin-walled  cells.  The  older  ones  are  complete  on  their 
inner  edge  already,  but  on  the  outer  are  still  in  the  proc- 
ess of  formation. 

It  is  supposed  that  from  the  point  f",  the  tissue  is  sec- 
ondary, produced  by  the  activity  of  the  cambium  ring. 
The  rind,  c/%  consists  of  roundish  cells.  Crystal  needles 
in  groups  or  in  bundles  are  found  in  the  cells  of  the  inner 
border  of  the  rind,  principall3\  These  so-called  "raph- 
ides  "  are  calcium  oxalate.  The  other  cells  of  the  rind 
contain  chlorophyll  grains.  The  bundles,  one  of  whose 
round  sections  are  seen  at  /'"'',  are  those  provided  for  the 
leaves.  The  stout  layer  of  thin-Avalled,  colorless,  radially- 
ai-ranged  cells,  I,  which  pass  outwardly  into  a  brown,  less 
regular  tissue  is  the  cork  layer ;  on  the  inside,  young, 
colorless,  and  on  the  outside,  old,  irregularly  elongated  and 
browned  cork-tissue. 

Stain  a  transverse  section  with  coralline.  The  bundles 
come  out  sharply.     The  lignified,  secondary,  fundamental 


94  CLOSED  VASCULAR  BUNDLES. 

tissue  is  stained  another  shade  of  red,  the  unlignified,  a 
pale  rose  red.  The  raphide  cells  seem  filled  with  a  red 
liquid.  So  we  know  that  the  raphides  are  embedded  in  a 
homoo^eneous  mucilaoe  which  absorbs  coralline.  This  re- 
ao;ent  stains  veo;etable  mucilao^e  :  neither  cold  nor  hot  alco- 
hoi  will  discolor  this  mucilage  thus  stained.  But  mucilage 
derived  from  cellulose  is  bleached  in  both  hot  and  cold  al- 
cohol (7).  In  this  we  have  our  test  of  starch  and  cellulose 
mucilage.  Gum  is  not  stained  by  coralline.  A  mixture 
of  cum.  and  mucilao'e  is  or  is  not,  accordins;  to  conditions.- 
An  aqueous  solution  of  nigrosin  will  not  stain  the  mucilage 
of  this  plant,  even  with  long  treatment,  but  it  will  that  of 
Rumex. 

This  survey  of  the  transection  shows  us  the  process  of 
the  radial  growth  of  the  plant.  We  will  omit  the  study 
of  details  and  of  the  longitudinal  section. 

Notes. 

(1)  In  regard  to  the  vascular  bundles  generally,  see  de  Bary,  Yergl. 
Anatomie  1877,  especially  Chapter  vni,  where  the  whole  of  tlie  older 
literature  maj'  be  found.  Numerous  critical  investigations  of  the  mor- 
phology of  the  vascular  bundles,  which  have  recently  appeared,  have 
not  had  a  coherent  treatment.  G.  Haberlandt,  on  the  contrary,  in  the 
Encyklopiidie  der  Naturwissenschaften,  Handbuch  der  Botanik.  Bd.  n, 
p.  593,  has  accomplished  this  in  part  by  attempting  a  phj-siological  in- 
terpretation of  morphological  facts,  in  his  physiologico-anatomical 
works. 

(2)  The  designation  vascular  part  and  sieve  part  was  introduced  by 
de  Bary,  Vergl.  Anat.,  p.  330. 

(3)  See  Haberlandt,  in  the  history  of  the  development  of  the  me- 
chanical tissue-systems  of  plants. 

(4)  Schwendener.  The  mechanical  principles  in  the  anatomical 
structure  of  the  mouocotyledons. 

(5)  This  staining  fluid  was  introduced  by  SzyszyloAvicz.  See  Bot. 
Centralbl.  Bd.  xii,  p.  138. 

(6)  See  Tangl,  Jahrb.  f.  wiss.  Bot.,  Bd.  xir,  p.  170. 

(7)  See  Szyszylowicz  at  the  same  place. 


LESSON  IX. 
Open  Collateral  Vascular  Bundles. 

For  our  study  of  the  open  collateral  bundles  of  the  di- 
cotyledons we  will  take  a  runner  of  Ranunculus  repens. 
Staiu  the  section  with  coralline.  We  tiud  the  bundles  is- 
olated from  each  other  and  arranged  in  a  simple, circle  in 
the  stem.  The  fundamental  tissue  consists  of  rounded  cells 
which  diminish  towards  the  surface  of  the  stem,  contain 
chlorophyll  and  have  large  intercellular  spaces  between 
them. 

At  the  surface  is  the  epidermis,  but  within,  the  stem  is 
hollow,  caused  by  the  separating  and  rupturing  of  the  cells. 
The  vascular  bundles  have  the  same  appearance  as  those 
of  the  monocotyledons,  the  same  parts  appearing  in  the 
same  order.  The  woody  part  consists  of  vessels  and  thin- 
walled  parenchyma  cells ;  the  ring  and  spiral  vessels  on 
the  innerside  of  the  bundle  next  the  vessels  take  up  but 
little  coloring  matter,  Fig.  44,  s.  The  other  subangular 
vessels  are  colored  brown-red.  In  these  walls  are  bor- 
dered pits,  m.  Between  these  vessels  lie  the  soft- walled 
primary  wood  parenchyma.  In  the  bast  is  again  the  alter- 
nation of  large  sieve-tubes  and  small  conducting  cells. 
But  the  bast  is  separated  from  the  woody  part  by  a  many- 
layered  stratum  of  radially-arranged  cells,  c.  This  ar- 
rangement betrays  their  cambium  origin.  A  cambium 
layer  separates  the  woody  from  the  bast  part,  and  distin- 
guishes this  from  the  monocotyledons.  The  activity  ot 
this  cambium  layer  is  indeed  limited  outwardly,  but  there 
is  enough  of  it  to  give  the  bundle  a  place  among  the  "open 
bundles,"  that  is,  those  capable  of  a  lateral  growth. 

(95) 


96 


OPEN    VASCULAR    BUNDLES. 


It  has  formed  a  stratum  several  layers  thick  of  thhi- 
walled  cells  and  then  ceased  to  grow.  The  bast  is  pro- 
tected oil  the  outside  by  a  cord  of  sclerenchyma  cells, 
colored  a  beautiful  coralliue  red.  So  also  the  iuner  edge 
of  the  buudle  is  iuclosed  by  another  but  thinner-walled 
layer  of  such  sheath  cells.  The  sheath  cells  do  not  meet 
at  the  sides  of  the  buudle. 


Fig.  44.  Transection  of  the  vascular  bxuidle  of  a  runner  of  Ranunculus  repens. 
s,  spiral  vessels;  m,  bordered  pitted  vessels ;c,  cambium;  v,  sieve-tubes;  vg,  sheath. 
Xiso. 

In  the  longitudhial  section,  ring,  spiral  and  pitted  ves- 
sels, and  between  them  elongated  wood  parenchyma  cells, 
are  easily  made  out.  Then  follow  thin-walled  cambium 
cells,  sieve-tubes,  conducting  cells,  and  finally  sheath  ele- 
ments with  but  slightly  inclined,  porous,  transverse  divi- 
sion walls. 


OPEN   VASCULAR   BUNDLES.  97 

The  vascular  bundles  of  celandine,  ühelidonium  majus, 
are  so  like  those  of  the  Ranunculus  repens  that  a  cross-sec- 
tion can  be  easily  understood  from  that.  We  prefer  alco- 
hol material.  The  wood  part  shows  large  vessels  pressed 
close  together  which  in  the  older  sterns  have  yellowish 
walls.  The  bast  is  strongly  developed  ;  between  the  two 
lies  the  stratum  of  thin-walled,  radially-arranged  cells 
produced  by  the  activity  of  the  cambium.  The  sheath 
appears  only  in  a  bundle  of  thick-walled  sclerenchyma 
cells  on  the  outer  border  of  the  bast,  in  old  stems  colored 
yellow.  Running  around,  just  within  the  epidermis,  and 
separated  from  it  b}^  a  cell-layer  two  cells  thick,  is  a  ring 
of  sclerenchyma  ceils,  like  those  which  protect  and  sup- 
port the  bundles,  making  a  common  sheath  to  the  whole 
inner  tissue  of  the  stem.  In  and  on  the  bundles  we  meet 
an  element  not  heretofore  seen,  the  milk-tubes.  They 
are  tilled  with  a  dark  brown  substance,  which  is  the  orange- 
red  milk-sap  of  the  plant  into  which  the  alcohol  has  run. 

They  are  found  in  the  bast  of  the  vascular  bundles,  also 
on  the  inner  bolder  of  the  wood  part ;  especially  numerous 
on  the  sides  of  the  bundles  and  the  outer  edge  of  the 
sclerenchyma  tissue,  and  scattered  singly  through  the  fun- 
damental tissue  between  the  bundles.  They  are  all  thin- 
walled,  even  those  intercalated  in  the  outer  edge  of  the 
sclerench3ma  tissue.  It  is  impossible  not  to  see  them. 
In  the  longitudinal  sections  they  are  easily  recognized  also 
by  their  yellow-brown  contents.  They  run  parallel  to  the 
axis  of  the  stem,  are  lurnished  with  transverse  walls  which 
are  perforated  with  one  or  more  pores,  and  yet  these  walls 
are  quite  lacking  sometimes  in  places  where  we  expect  to 
tind  them.  Often  some  of  the  vessels  in  the  bundles  are 
found  filled  with  coagulated  milk-sap. 

Stain  a  transverse  section  with  coralline,  then  ap[)ly  a 
drop  of  potash  \ye  to  the  edge  of  the  cover-glass.     The 


98  OPEN    VASCULAR    BUNDLES. 

vessels  will  appear  fuchsiu-red,  the  sclerenchj'ina  cells 
rose-red,  while  the  milk-tubes  and  their  dark  brown  con- 
tents will  come  out  ver}"  distiuctly.  Put  a  very  thin  lon- 
gitudinal section  in  45  per  cent  acetic  acid  carmine,  and 
nuclei  may  be  detected  in  the  milk-tubes,  but  not  very 
easily.  Lateral  unions  of  the  milk-tubes  have  not  been 
seen  in  this  plant. 

Aristolochia  siplio,  or  Dutchman's  pipe,  affords  an  un- 
commonly good  object  in  which  to  study  the  lateral  growth 
of  the  dicotyledons.  Make  a  section  of  a  branch  3  to  4 
mm.  thick.  AVith  a  lens  observe  the  iuner  loose  pith; 
about  this  a  circle  of  isolated  vascular  bundles  ;  about  this 
a  contiuuous  white  ring;  then  green  rind-tissue  ;  finally,  a 
yellowish-green  peripheral  envelope.  With  a  low  power 
under  the  microscope  we  see  that  the  pith  is  composed  of 
large  round  cells  in  part  filled  with  air.  In  the  vascular 
buudles  the  wood  part  is  darker  and  much  broken  by  the 
wide  cavities  of  the  vessels.  The  cambium  zone  follows, 
composed  of  narrow,  radially-arranged  light  cells,  and  here- 
upon the  bast  cells,  not  so  light  nor  so  regularly  arranged 
but  much  larger.  Each  bundle  is  bordered  about,  espec- 
ially on  its  outer  part,  by  parenchymatous  tissue  contain- 
ing some  chlorophyll  grains,  aud  eventually  also  reserve 
substances.  The  white  ring  beyond  is  formed  of  much 
thickened  sclerenchyma  cells  which  project  inward  some- 
what in  wedge-shaped  masses  between  the  vascular  buu- 
dles. Abutting  upon  this  ring  is  the  tissue  which  contains 
chlorophyll  and  air-filled  intercellular  spaces.  Beyond 
this  comes  narrow-celled  tissue  containing  chlorophyll,  the 
cell-walls  white  and  thickened  at  the  corners,  on  account 
of  which  they  are  called  "  collenchyma  "  cells.  Finally, 
the  epidermis. 

Now,  make  a  very  thin  section  of  a  bundle  from  alco- 
hol material,  that  has  previously  lain  iu  a  mixture  of  equal 


OPEN   VASCULAR   BUNDLES. 


99 


parts  alcohol  and  glycerine.  Stain,  by  immersing  for  some 
time  in  coralline.  Fig.  45  gives  a  representation  of  a  sec- 
tion of  a  bundle  made  from  a  growing,  this  year's  branch, 
about  the  besrinnino'  of  June.  The  vascular  bundle  bemns 
on  the  inner  edge  with  thin-wallcd,  primary  wood  paren- 


Fig.  45.  Transection  through  a  young  twig  of  this  year's  growth  of  Aristolochia 
siplio,  showing  a  vascular  bundle  after  the  beginning  of  the  active  growth  of  the 
cambium,  j:?,  parenchymatous  elements  on  the  inner  edge  of  tlie  wood  part;  m'  and 
m",  vessels  with  bordered  pits;  ic,  interfascicular  cambium  extending  into  the  fas- 
cicular cambium,  that  is,  into  the  cambium  within  the  vascular  bundle;  v.  sieve- 
tubes;  c,  rind-parenchyma;  sk,  inner  part  of  the  ring  of  sclerenchyma  fibres.  X  130- 

chynia,^,  in  which  narrow  vessels,  which  gradually  become 
wider  (protoxylem  elements),  are  inclosed,  the  cells  them- 
selves also  becoming  thicker-walled.  The  same  also  may 
be  said  of  the  vessels,  while  the  intervening  spaces  are 


100  OPEN    VASCULAR    BUNDLLS. 

taken  up  by  tracheids,  with  thickened  and  border-pitted 
walls.  The  thick-walled  wood  parenchyma,  vessels,  and 
tracheids  take  an  intense  red,  while  the  thin- walled  are  col- 
ored a  faint  rose.  The  two  largest  vessels  are  seen  in  the 
process  of  development.  Between  these  lies  thjn-walled, 
serially-arranged,  secondary  tissue,  of  cambium  origin. 
On  the  outer  border  of  the  two  large  vessels  is  the  cam- 
bium zone,  in  which  an  especially  flat,  not  very  sharply 
defined  cell-layer  represents  the  initial  stratum.  Follow- 
ing this,  are  like  cells  of  the  bast,  the  radial  arrangement 
of  which  betrays  their  secondary  cambium  oiigin.  In  the 
middle  of  the  bast  are  the  sieve-tubes  from  which  the  ac- 
companying conducting  cells  are  distinguished  by  the  con- 
tents existing  in  a  majority  of  them.  The  outer  part  of 
the  bast,  the  protophloem,  is  taken  up  by  narrower  sieve- 
tubes,  which  are,  therefore,  not  so  sharply  contrasted  wdth 
the  conducting  cells.  The  bast  is  separated  from  the  sole- 
renchyma  ring,  sk,  by  large-celled  rind-parenchyma.  The 
sclerenchyma  is  as  intensely  colored  as  the  wood  part  of 
the  vascular  bundle.  The  protophloem  elements  are  com- 
pressed by  the  multiplication  and  growth  of  the  new  cells 
from  the  cambium.  Such  a  section  shows  very  perfectly 
the  formation  of  the  interfascicular  cambium. 

With  the  beginning  of  the  activity  of  the  cambium  in 
the  vascular  bundle,  the  cells  of  the  fundamental  tissue 
adjoining  the  same  at  the  sides  also  increase  by  self-divi- 
sion through  the  introduction  of  dividing  w\alls,  ic.  So 
the  elements  of  the  fundamental  tissue  are  formed  into  a 
cambium  stripe  which  unites  the  cambium  layer  in  the  cir- 
cularly placed  vascular  bundle  into  a  contuiuous  cambium 
ring.  As  the  figure  shows,  it  is  very  easy  to  follow  the 
formation  of  the  interfascicular  cambium  in  this  plant,  and 
to  recognize  for  a  long  distance  the  original  outline  of  the 
divided  fundamental-tissue  cells.  The  sheath  is  altooether 
lacking  in  this  plant  about  the  single  vascular  bundles. 


OPEN    VASCULAR    BUNDLES.  101 

The  sclerenchyma  ring  forms  a  common  sheath  about  the 
whole  inner  tissue  of  the  stem.  A  radial  lonrntudinal  sec- 
tion  cut  through  the  exact  middle  of  a  vascular  bundle 
and  stained  with  coralline  shows,  on  the  innermost  side, 
elongated  primary  wood  parenchyma  with  transverse  divi- 
sion walls,  between  which  are  very  narrow,  somewhat 
compressed  ring  vessels  ;  then  somewhat  wider  ring  vessels, 
which  in  part  pass  into  spiral  vessels  ;  then  closely  spiralled 
wider  vessels  which  pass  into  reticulated  vessels ;  finally, 
the  enlarged  border-pitted  vessels.  Between  these  vessels 
are  much  elongated,  border-pitted,  empty  tracheids  ;  sin- 
gle fibre  cells,  like  the  tracheids  in  ftn-m,  but  having  un- 
bordered  pits  and  filled  with  starch  ;  thick-walled  wood 
parenchyma,  shorter,  with  transverse  walls,  likewise  un- 
bordered  pits  and  starch.  The  incomplete,  large  vessels 
are  still  wide,  cylindrical,  thin-walled  cells,  separated  by 
transverse  division  walls,  with  rich  protoplasmic  wall-lin- 
ing, and  a  nucleus.  In  the  fully  completed  vessels,  noth- 
ing of  their  cell-contents  is  to  be  found,  and  of  the  division 
wall,  in  the  pitted  vessels,  there  remains  only  the  ring-like 
projecting  diaphragm.  The  flat  cells  of  the  caiiibium  zone 
are  rich  in  protoplasmic  contents,  nucleus  and  delicate 
transverse  division  walls.  The  sieve-plates  are  quite  extra- 
ordinarily beautiful.  When  they  are  somewhat  inclined, 
the}'  present  to  the  ol)server  their  whole  rosy  surface  with 
dark,  sparkling  points.  In  those  sieve-plates  which  are 
much  inclined,  the  plate  is  divided  by  clear  belts  without 
pores,  into  several  rose-colored,  dotted  and  superimposed 
sections.  The  side  walls  of  the  sieve-tubes  are  also  cov- 
ered with  small,  transversely  extended,  finely  dotted,  rose 
colored  sieve-pits.  In  the  periphery  of  the  bast  occurs, 
in  the  most  astonishing  manner,  the  formation  of  the  cal- 
lus-plates, those  bright,  rose-colored,  strongly  refractive 
masses,  which  are  rounded  upon  the  free  side,  and  which 


102  OPEN   VASCULAR   BUNDLES. 

cover  both  sides  of  the  sieve-pl;ite  in  like  mass,  or  are 
prepoiideratingly  on  one  side  only.  The  sieve-pits  on  the 
lateral  walls  also  have  minute  callus-plates.  We  also  find 
here  with  the  sieve-tubes,  the  narrower  conduct! ng-cells 
tilled  with  their  conteuts.  The  bast  is  separated  from  the 
sclerenchyma  ring  by  the  broader  —  and,  as  this. section 
shows,  also  relatively  shorter  —  pareuchyma  cells.  The 
sclerenchyma  fibres  of  the  ring  are  very  long,  pointed  at 
their  ends,  comb-like  with  their  ends  interlocked  and  pro- 
vided with  pores.  And,  finally,  we  notice  that  the  coUen- 
chyma  cells  bordering  on  the  epidermis,  are  several  times 
longer  than  broad  and  are  joined  with  transverse  walls. 

Now  cut  a  section  from  an  older  branch,  say  one  10  mm. 
in  diameter  and  examine  it  with  the  lens.  The  pith  and 
the  medullary  rays  are  white,  the  wood-bodies  yellowish. 

The  thickest  medullary  rays,  some  ten  or  twelve  in  num- 
ber, open  into  the  pith,  and  are  those  primary  rays  which 
in  the  beginning  separated  the  vascular  bundles.  The  old- 
est wood  part  of  the  bundles  border  on  the  pith.  Since 
the  wide  vessels  are  lacking  in  them,  they  seem  to  be  a 
thicker,  darker  ring  penetrated  by  the  primary  medullary 
rays.  To  these  succeed  the  concentric  yearly  rings.  The 
width  of  the  vessels  increases  in  the  first  year's  growth  till 
it  reaches  a  definite  greatest  diameter.  The  boundary 
of  the  yearly  ring  is  clearly  marked  by  the  larger  vessels, 
since  those  of  widest  cavity  are  produced  in  the  begin- 
ning of  the  development  in  the  spring.  The  outer  part 
of  the  yearly  ring  contains  no  vessels  distinguishable  with 
the  lens.  As  the  secondary  Wood-bodies  increase  in  cir- 
cumference, new  medullary  rays  are  intercalated  Avhich  we 
designate  as  secondary  rays  of  the  second,  third,  fourth^ 
etc.,  orders.  The  intercalation  of  secondary  rays  follows 
with  the  greatest  regularity.  The  farther  we  go  from  the 
centre,  the  more  numerous  are  the  medulhuy  rays  and  the 


OPEN    VASCULAR    BUNDLES.  103 

shorter  are  the  newly  adclecl  ones.  On  the  onter  border  of 
the  wood  bod}^  we  see  the  caniljium  ring  as  a  dark  circle, 
the  medullary  rays  within  which  are  indicated  l^y  delicate 
lines.  Before  the  secondary  wood  parts,  Ave  see  the  clear 
brown-colored  secondaiy  bast  lying,  formed  from  succes- 
sive growths.  The  medulhiry  rays  extend  be3'ond  the  cam- 
bium in  consequence  of  its  supplementary  lateral  growth 
caused  b}^  the  increase  of  the  thickness  of  the  stem.  The 
bast  is  not  capable  of  this  supplementary  lateral  growth 
and  appears  thence  from  the  outside  to  be  nari'owed  and 
rounded.  The  original  continuous  sclerenchj'ma  ring  is 
dispersed  in  single  olive-green  colored  pieces  of  unlike 
size ;  likewise,  also  the  original  continuous  darker  olive- 
green  collenchyma  layer.  The  periderm  now  undertakes 
the  protection  of  the  interior,  and  as  a  brown,  distinctly 
laminated  sheath  covers  the  surface  of  the  stem.  The 
whole  of  that  part  subsequently  produced  by  the  cam- 
bium, which  includes  the  secondary  bast  and  the  extended 
medullary  rays,  becomes  secondary  rind,  which  confronts 
the  primary  rind  previously  existing  before  the  beginning 
of  the  lateral  growth  of  the  stem.  No  sharp  boundary 
exists  between  the  primary  and  secondary  rinds. 

Now  apply  a  higher  power  to  the  investigation  of  a  thin 
cross-section  of  this  stem.  The  pith  tissue  is  unchanged 
from  its  young  state,  only  that  it  has  numerous  crystal 
masses  of  calcium  oxalate.  The  primary  wood  parts  project 
into  the  pith  tissue,  formingthe  so-called "medulhuy  crown" 
or  "medullary  sheath."  The  hand-lens  will  not  show  this, 
as  the  inner  parts  arec(miposed  of  thin-walled  compressed 
cells.  First  on  entering  the  solid  part  we  find  the  wood-bod- 
ies clearly  marked  off  from  the  large  pitted  vessels.  The 
vascular  bundles  increase  in  breadth  correspondingly  to  the 
narrowing  of  the  medullary  rays.  The  vessels  formed  in 
the  spring,  up  to  the  third  or  fourth  annual  ring,  show  an 


104  OPEN    VASCULAR    BUNDLES. 

increase  in  volume.  From  the  spring  toward  the  fall,  the 
width  of  the  vessels  rapidly  decreases  in  each  annual  ring. 
Shortly  before  the  close  of  the  year's  growth,  only  very 
narrow  vessels  are  produced.  The  great  mass  of  the  wood 
consists  of  tracheids,  narrow,  thickened,  empty,  border- 
pitted  cells.  They  contain  air  or  water.  If  starch  grains 
are  ever  seen  in  them,  the  knife  has  carried  them  there 
from  neighl)oring  cells.  Distributed  about  the  circumfer- 
ence of  the  vessels  mainly,  but  also  among  the  tracheids, 
are  thinner-walled  cells  with  protoplasmic  contents  ;  also, 
commonly,  starch  and  flat  pits.  These  are  wood-paren- 
chyma and  fibre  cells.  The  vessels  are  provided  Avith 
bordered  pits,  only  when  they  touch  each  other  or  the 
tracheids.  When  a  vascular-pit  or  a  tracheid-pit  meets 
the  pit  of  a  wood-parenchyma  or  fibre  cell,  it  is  one-sided, 
that  is,  bordered  only  on  the  side  of  the  vessel  or  tra- 
cheid,  or,  so  to  say,  narrowed  in  its  opening  only  on  this 
side. 

The  closing  membrane  of  such  one-sided  pits  is  without 
central  thickening  (torus)  and,  unlike  such  thickened  mem- 
brane, is  colored  blue  with  chloriodide  of  zinc  (1). 

The  cells  of  the  medullary  rays  are  radiall}^  extended, 
relatively  thin-walled,  and  have  numerous  pores.  On  the 
outer  border  of  the  wood  substance,  we  easily  recognize 
the  cambium  formed  from  flat,  thin-walled,  radially-ar- 
ranged cells,  and  beyond  that  the  thin-walled  bjist.  Be- 
sides sieve-tubes  and  conducting-cells,  we  find  in  this  also 
starch-bearing  bast  parenchyma. 

The  secondary  bast  produced  by  the  cambium  has  con- 
sequently gained  the  latter  additional  elements.  With 
an  extremely  delicate  section  one  can  follow  in  the  bast 
the  alternation  of  uncompressed  with  fully  compressed 
cell  layers.  Similarly,  flatly  compressed  elements  have 
already  been  seen  in  the  one-year-old   branches  on  the 


OPEN   VASCULAR   BUNDLES.  105 

periphery  of  the  primary  bast,  the  appearance  repeating 
itself  consequently  in  the  bast  growth  of  subsequent 
years.  These  bands  of  flatly  compressed  cells  are  after- 
wards broken  into  parts  which  always,  and  after  a  time 
more  distinctly,  take  the  form  of  a  bow.  By  the  inter- 
calation of  new  medullary  rays  the  bast  is  constantly 
being  divided  so  that  every  outer  bast  part  spans  two  in- 
ner. Outside  of  the  sieve  parts  in  the  rind  are  the  sep- 
arated pieces  of  the  ring  of  sclerenchyma  fibres.  The 
pieces  are  separated  by  parenchymatous  tissue.  The  ring 
has  been  radially  broken  in  consequence  of  the  progres- 
sively lateral  growth  of  the  cambium,  and  the  adjoining 
tissue  of  the  rind  has  pushed  itself  in.  The  collenchyma 
ring  also  is  distributed  in  parts.  Still,  there  is  no  essential 
breaking  of  it  up;  rather,  in  single  places,  a  tangential  ex- 
tension of  the  cells  takes  place  which  then  in  parting  came 
in  and  so  the  parenchymatous  tissue  masses  get  their  ori- 
gin. The  surface  of  the  stem  is  covered  with  the  periderm 
which  presents  the  beautiful  alternation  of  broad  zones  of 
wide  thin-walled,  and  narrow  zones  of  small,  thick-walled 
cork  cells.  As  in  the  pith  and  medullary  rays  so  in  the 
rind  are  found  scattered  crystal  masses  of  calcium  oxalate. 
The  radial  longitudinal  section  shows,  in  the  first  place, 
the  wide  and  narrow  vessels,  border-pitted,  with  annular 
diaphragms ;  the  border-pitted  tracheids ;  the  fibre  cells, 
shallow-pitted  and  with  cell  contents ;  the  wood-paren- 
chyma cells,  likewise  with  cell  contents,  with  flat  pits, 
shorter,  less  thickened  than  the  tracheids  and  joined  together 
in  continuous  threads.  If  the  medullary  rays  have  been 
hit,  radial  lines  of  their  thin-walled  cells  will  be  seen  run- 
ning across  the  section.  On  the  outer  border  of  the  wood, 
we  recognize  the  cambium  cells,  rich  in  contents,  thin- 
walled  with  transverse  walls  between  ;  then  the  still  active 
l)ast,  and  here,  upon  the  flat  cells  of  the  older  bast,  the 


106  OPEN   VASCULAR   BUNDLES. 

compressed  alternating  with  the  uncompressed  parts.  The 
laminated  periderm  shows  up  very  beautifully  in  the  mar- 
gin of  the  section.  The  longitudinal  section  of  this  layer 
is  exactly  like  the  transverse  section,  the  cells  being  of 
the  same  breadth  or  height.  By  the  cutting  of  the  wood 
the  exact  course  of  the  medullary  rays  is  apparent  to  the 
unaided  eye.  This  comes  from  the  considerable  length  of 
the  internode  within  which  both  the  vascular  bundles  and 
the  medullary  rays  retain  their  direction  unchanged.  The 
tangential  section  shows  us  under  the  microscope,  the  med- 
ullary rays  in  the  form  of  broader  or  narrower  stripes  more 
or  less  parallel  to  each  other  separated  by  corresponding 
stripes  of  wood. 

As  it  is  not  a  little  difficult  always  to  distinguish  the 
different  tissues  in  the  complicated  image  shown  by  sec- 
tions of  the  wood,  we  will  try  another  method,  viz.,  that 
of  maceration.  For  this  purpose  take  a  wide  test-tube 
and  over  some  fragments  of  potassium  chlorate  pour  enough 
nitric  acid  to  cover  the  pieces  fully.  Put  into  this  a  not 
too  thin  secti(m  of  the  wood  and  heat  to  boiling  over  a 
flame.  Let  it  stand  for  some  minutes  and  pour  the  whole 
into  a  laro;er  dish  of  water.  With  the  glass  rod  remove 
the  floating  section  into  another  dish  of  water  and  thence 
into  a  drop  upon  the  slide.  This  experiment  should  not 
be  made  in  the  same  room  with  the  microscope  else  the 
gas  may  damage  the  instrument.  The  section  should  now 
be  disintegrated  with  needles  so  as  to  have  its  elements 
separated.  If  the  reagent  has  properly  worked,  the  mid- 
dle lamella  will  be  dissolved  and  the  cells  will  easily  come 
apart.  Now  we  shall  find  all  of  the  elements,  heretofore 
studied  in  connection,  entirely  isolated.  They  are  mostly 
well  preserved,  only  that  they  have  been  robbed  of  their 
wood  substance  and  will  be  stained  violet  for  the  most  part 
with  chloriodide  of  zinc.     First  of  all  we  shall  see  the  pit- 


OPEN  VASCULAR  BUNDLES.  '         107 

ted  vessels  mostly  sepurated  into  pieces  corresponding  to 
the  annular  diaphragms. 

The  tracheids  are  especially  numerous  with  attenuated 
rounded  ends  and  bordered  pits.  These  pits  present  them- 
selves now  in  the  smaller  walls  as  narrow  oblique  slits. 
But  by  proper  focussing  it  is  always  possible  to  demon- 
strate that  they  widen  outwardly.  When  some  of  the  tra- 
cheids are  found  still  joined  together,  the  pits  appear  in 
the  form  of  a  cross,  the  corresponding  pits  on  the  two  ad- 
jacent cells  being  inclined  in  opposite  directions.  Besides 
vessels  and  tracheids  are  thin-walled  wood-parenchyma 
cells  with  large  flat  pits.  They  are  also  recognizable  by 
their  compacted  and  knotty  cell  contents.  We  find  also 
isolated  forms  which  are  like  those  of  the  fibre-cells,  occa- 
sionally with  but  one  cell  cavity  but  commonly  divided 
into  several  shorter  parts  one  above  the  other  by  transverse 
or  oblique  walls.  Those  with  a  single  cavity  are  what 
we  have  heretofore  known  as  fibre-cells,  but  which  may 
be  better  known  as  "substitute  fibre-cells"  since  they  re- 
place the  wood-parenchyma  cells. 

The  compound  forms  which  together  replace  the  wood- 
parenchyma  are  apparentl}^  produced  by  the  division  of  a 
single  mother-cell.  The  transverse  division  walls  must 
have  been  formed  at  an  early  period  when  the  mother-cell 
w^as  still  thin-walled,  for  they  show  the  same  thickness  and 
the  same  pits  as  the  side  walls  and  must  therefore  have 
been  thickened  at  the  same  time  with  these. 

NOTK. 

(1)     See  Rnssow,  Bot.  Ceiitralbl.    Bd.  xiii,  p.  140. 


LESSON  X. 
Structure  of  the  Coniferous  Stems. 

Wn  shall  now  undertake  the  thorough  study  of  the 
structure  of  the  stems  of  the  fir  tree,  Pinus  sylvestris. 
We  shall  find  the  lateral  growth  entirely  different  from 
that  of  the  Aristolochia.  In  the  pine  the  secondary  growth 
of  the  wood  consists  entirely  of  the  formation  of  one  ele- 
ment, the  tracheids.  Vessels  are  fonnd  in  the  pine,  only 
in  the  medullary  sheath,  in  the  primary  wood  of  the  vas- 
cular bundles.  A  transection  shows  that  the  inner  edges 
of  the  dark-colored  wood  which  projects  into  the  pith  con- 
sist of  narrow  elements  with  somewhat  brown  walls.  A 
thin  longitudinal  section  shows  them  to  be  spiral  vessels. 
Some  such  vessels,  which  likewise  have  bordered  pits  and 
spiral  bands,  constitute  a  transitional  form  to  the  trache- 
ids with  bordered  pits  o\\\j. 

We  will  use  alcohol  material  in  making  a  section  of  the 
cambium,  the  fresh  being  liable  to  tear,  and  the  dry  diffi- 
cult to  cut.  La}^  the  wood  about  twenty-four  hours  in  a 
mixture  of  equal  parts  alcohol  and  glycerine  before  cut- 
ting. Alcohol  material  has  the  advantage  of  having  the 
cell-contents  fixed  also.  The  wood  should  be  cut  in  June 
or  July  when  the  cambium  is  in  full  activity  and  put  into 
alcohol. 

Make  the  section  from  the  periphery  of  a  good  sized 
stem,  as  the  tracheids  in  the  later  annual  rings  are  larger. 
We  will  examine  the  section  in  glycerine.  But  in  case  we 
use  reagents  with  it  we  shall  previously  wash  it  Avitli  wa- 

(108) 


STRUCTURE  OF  CONIFEROUS  STEMS.  109 

ter.  We  begin  by  making  a  section  of  the  stem  from  the 
periphery  inward  across  several  of  the  annual  rings,  the 
cambium  and  the  rind. 

We  see  the  tracheids  arranged  in  a  radial  series  and 
occasionally  a  row  is  doubled.  These  elements  are  quad- 
rangular ;  sometimes  five-and*six-angled.  In  the  fall  the 
Avails  become  thicker  and  the  tracheid  narrower.  Imme- 
diately adjoining  these  are  the  wider  and  thinner-walled 
ceHs  of  the  following  spring,  thus  distinctly  marking  even 
to  the  naked  eye  the  limit  of  the  year's  growth.  Parallel 
to  the  radial  rows  of  tracheids  are  the  medullary  rays,  nar- 
row and  of  one  layer  of  cells,  seldom  of  more,  distin- 
guished by  their  cell-contents  of  starch.  On  the  radial 
walls  of  the  tracheids  stand  the  bordered  pits  whose 
structure  we  already  know.  Between  the  tracheids  and 
the  medullary-ray  cells  are  very  wide,  half- bordered,  or 
one-sided  pits,  so  wide  thit  they  cover  almost  the  whole 
breadth  of  the  wall  of  the  tracheids.  They  must  be  called 
one-sided  because  the  border  is  developed  only  in  the 
tracheid.  The  closing  membrane  is  bent  forward  in  the 
tracheid  and  has  no  torus.  Treated  with  chloriodide  of 
zinc  the  closing  membrane  colors  blue  (1),  while  it  re- 
mains uncolored  in  the  two-sided  bordered  pits.  The  cells 
of  the  medullary  rays  at  those  points  where  they  are  touched 
b}^  the  tangential  walls  of  the  tracheids  are  provided  with 
a  thickened  ledge.  (See  the  medullary  ray  m,  Fig.  47, 
and  the  tracheids  bearing  on  it.) 

If  the  section  shall  strike  a  zone  in  which  the  cells  of 
the  medullary  rays  are  empty,  we  shall  find  them  united 
to  the  adjoining  tracheids  with  two-sided  bordered  pits. 
In  the  immediate  neighborhood  of  the  cambium  we  see 
the  incomplete  tracheids  of  the  young  wood.  The  walls 
of  the  cells  increase  rapidly  in  thickness  toward  the  cam- 
bium zone.  In  sections  from  much  older  stems  we  see 
the  radial  walls  within  the   cambium  zone  again  become 


no 


STRUCTURE  OF  CONIFEROUS  STEMS. 


Fig.  46.-  Part  of  a  transection  of  an  old  stem 
of  Pinus  sylvestris.  The  section  crosses  the 
cambium  (i,  initial  layer)  and  ends  on  the  one 
side  in  the  young  wood  and  on  the  other  in  the 
young  bast.  1,  2,  3,  developmental  stages  of 
bordered  pits ;  vi.  medullary  ray ;  c,sieve  plate ; 
/.•,  flat  cells  with  brown  contents  afterwards 
bearing  crystals.  X  510- 


thicker  (2).  See  Fig. 
4(i.  Tliat  which  we 
must  cull  the  cambium 
consists  of  an  initial 
layer  theoretically  jone- 
cell  thick,  ^,  which  by 
continuous  tangential 
division  furnishes  the 
tissue  mother-cells  on 
both  the  wood  and  bast 
sides,  and  out  of  these, 
by  the  division  of  the 
mother-cells,  the  wood 
and  bast  have  their 
origin.  No  distinct 
boundary  can  be  drawn 
between  the  initial  layer 
and  the  tissue  mother- 
cells  of  bast  and  wood 
on  each  side.  The 
youngest  partition  walls 
are  sharply  joined  to  the 
radial  side  walls,  i. 
Somewhat  older  parti- 
tion walls  are,  on  the 
contrary,  a  little  thick- 
ened at  the  points  of 
juncture.  Upon  the 
wood  side  the  develop- 
ment of  the  bordered 
pit  may  be  followed  (1, 
2,  3).  The  series  of 
tracheids  is  continued 
through  the  cambium 
into     a    row    of    bast 


STRUCTURE  OF  CONIFEROUS  STEMS.         Ill 

cells  which  maintain  the  radial  arrangement  quite  as  fully. 
The  cell  walls  thicken  rapidly  on  the  liast  side,  but  are 
of  a  duller  white  and  less  sparkling  than  the  wood  cells. 
On  the  radial  walls  of  the  wide  bast  cells  are  sieve-plates, 
corresponding  to  the  places  occupied  by  bordered  pits  in 
the  wood.  In  very  thin  sections  they  may  be  recognized 
by  fine  pores  which  penetrate  these  spots.  Mainly,  bands 
of  single  flattened  cells  alternate  with  the  thicker  layers 
of  sieve-tubes.  These  bands  represent  the  bast  paren- 
chyma. The  majority  of  these  cells  are  indicated  by 
their  strongly  refractive  brown  contents,  k.  In  cells  far- 
ther removed  from  the  cambium,  one  or  two  crystals 
may  be  seen  in  the  brown  substance.  Since  in  this  tree 
but  one  bast  parenchyma  band  is  formed  in  the  whole 
year,  these  may  be  used  for  determining  the  age  of  the 
bast  part.  Between  the  cells  with  crystals  lie  those  tilled 
with  starch.  So  between  the  sieve-tubes  are  distributed 
starch  cells  and  crystal  cells,  singly  or  in  numbers.  Med- 
ullary rays  continue  from  the  wood  through  the  cambium 
into  the  bast,  and  in  the  latter  a  part  of  its  cells  contain 
starch.  Only  a  comparatively  narrow  zone  of  the  bast 
will  keep  the  original  arrangement  taken  by  the  elements. 
Beyond  that  zone  the  radial  series  is  bent,  the  cell  walls 
begin  to  be  browned,  the  cell  cavity  more  or  less  com- 
pressed together  so  that  the  radial  walls  appear  bent  and 
wavy.  Only  those  cells  of  the  bast  and*  the  medullary 
rays  which  contain  starch  are  rounded  out*  and  full.  Fi- 
nall^^  the  sieve-tubes  and  crystal- bearing  ^cells  are  quite 
compressed  and  tangentially  extended,  and  like  a  lami- 
nated membrane  separate  the  large  starch-bearing  cells. 
The  outer  rind  now  seems  to  consist  entirely  of  the  lat- 
ter cells.  Farther  towards  the  outside  of  the  rind  one 
comes  to  small  leaves  of  cork  and  from  these  deeply 
browned  dead  tissue  is  tangentially  separated. 

The  resin-ducts  have  not  been  mentioned.     Every  sec- 


112         STRUCTURE  OF  CONIFEROUS  STEMS. 

tioii  of  the  wood  ^hows  them.  But  in  the  alcohol  prepara- 
tion they  have  lost  their  resin  contents,  and  consequently 
show  their  structure  all  the  better.  In  a  transection  it 
appears  as  an  intercellular  passage,  Fig.  47,  ^,  surrounded 
by  a  layer  of  large  thin-walled  cells,  e.  The  walls  of  these 
cells  are  browned,  have  a  nucleus,  and  a  wall  layer  of  proto- 
plasm. Bordering  on  these  is  a  second  layer,  flatter,  and 
poorer  in  cell  contents,  then  a  more  or  less  perfect,  and 
•^  indeed  here  and  there  a  double 

Ji-j-.'L-Wr^f /< ,,     tjß,        layer  of    large   starch-bearins: 

ji?  n  H:  jL~^^^:=;^--^^:^i      cells,  a.       The   latter  will  be 

^v  ;  ^^    ':_i4 -^^fe^^lK  —      surrounded  by  tracheids,  and 

_J^|?^^^'j?-'jF|j^'jv..-|^  will  eventually  rest  against  a 

Ji^'f^  g  /«;  C  ^^o\ '^  medullary  ray.  Conjunction 
~^Z;*^-T  =•' -^  /■  •  .  J  fe  with  one  such  is  generally  de- 
_^y;;,  ^         .    '    '  l_  sirable    for  each  resin- duct  at 

V-'4;(y,CC::l  /«^^^  some  one    place.     The  resin- 

I^JW^^-^II     if  duct  is  produced,  as  their  life- 


^   "'''^^n^'^i  ''■*  history  shows,  by  the  drawing 

Fig.  47.  Resin-duct  from  the  wood     apart     of    Certain    coutlguous 

€)f  Piiiuf  sylvestris,  i,  the  (\\\ct  üUeiX 

with  resin;  e,  the  cells  surrounding       CellS. 

theduct;«,  starch-bearing  cells;«,  ^y^     ^y-^    ^^^^^^    j^j.^j.^    ^    ^q^_ 

trachiids,  m,  medullary   lay  cells. 

X240.  tion  of  a  fre.sh  stem,  and  find 

that  the  passage  is  filled  with  resin.  It  is  very  refractive 
and  takes  the  form  of  irregularly-shaped  drops.  Alcohol 
causes  them  to  disappear.  Alcanna  tincture  colors  them 
as  it  does  oil  drops.  Upon  the  section  on  the  slide  in  a 
drop  of  water,  lay  a  thin  shaving  of  the  bark  of  a  dry  al- 
canna root.  Put  on  the  cover-glass,  and  add  50%  alco- 
hol, and  let  it  stand  for  an  hour.  Then  remove  the 
alcanna,  and  it  will  be  found  that  the  resin  elements  are 
stained  a  beautiful,  dark  red,  while  the  other  parts  of  the 
section  remain  colorless.* 

*It  is  much  more  convenient  to  use  alcanna  extract,  which  may  be  had  of  most 
aiJothecavies,  certainly  of  all  dealers  in  microscopical  goods. — A.  B.  H  . 


STRUCTURE  OF  CONIFEROUS  STEJMS.  113 

Chloriodide  of  zinc  colors  the  trache'id  walls  of  alcohol- 
material  sections  yellow-brown;  the  innermost  thickening 
layer  which  touches  the  boundary  membrane  is,  in  part, 
colored  violet.  Protoplasmic  contents  and  nuclei  are  eas- 
ily seen  in  the  imperfectly  developed  tracheids  near  the 
cambium.  Thus  it  is  very  easy  to  demonstrate  that  the 
fully  developed  tracheids  have  lost  all  their  living  con- 
tents. The  cambium,  with  the  3'oungest  adjoining  cells,  is 
stained  a  light  violet,  the  older  bast  walls  a  dark  violet. 
The  contents  of  the  crystal-bearing  cells  remain  brown, 
the  cells  of  the  periderm  red-brown.  The  thin-walled  cells 
which  surround  the  inner  surface  of  the  resin-duct  are  col- 
ored a  dull  violet. 

By  staining  a  section  made  through  the  cambium  with 
coralline,  we  easily  observe  the  gradual  extinction  of  the 
lignin  in  the  cell  walls  in  the  neighborhood  of  the  cambium, 
the  coralline  coloring  the  lignified  differently  from  the  un- 
lignified  membrane.  Lay  the  section  for  some  time  in 
coralline  soda,  and  then  examine  in  glycerine.  The  lig- 
nified membranes  are  colored  an  intense  red,  but  losing 
that  gradually  towards  the  cambium,  the  color  changes 
from  a  red  to  a  pale  yellow.  The  bast  has  the  cell  walls  a 
pale,  reddish-yellow,  the  sieve-plates  a  pronounced  rose, 
especially  where  they  are  covered  with  the  callus  masses, 
and  the  starch  grains  being  stained  a  rose  color  bring  this 
tint  into  prominence  in  the. outer  bast. 

Now,  make  a  radial  section  again  from  the  alcohol 
material,  and  the  tracheids  pointed,  interlocked,  border- 
pitted,  are  seen  as  before.  The  superficial  view  of  the 
bordered  pit  is  well  known.  The  bordered  pits  are  small 
and  infrequent  in  the  tracheids  formed  in  the  autumn. 
The  medullary  rays  run  across  the  tracheids.  The  rays 
sometimes  occur  sixteen  cells  high  but  are  usually  much 
less.     They  consist  (4)  of  radially-extended,  serially~ar- 

8 


114  STRUCTUKE  OF  CONIFEROUS  STEMS. 

ranged  cells,  the  middle  ones  having  starch,  and  on  their 
broad  sides,  next  the  tracheids,  showing  one-sided  bor- 
dered pits.     The  upper  and  under  three  rows  of  cells  are 
empty,  with  small  bordered  pits.    In  this  respect  they  agree 
somewhat  in  structure  and  behavior  with  tracheids,  and 
might  be  so  named,  but  we  prefer  to  limit  this  term  to  the 
elements  in  the  wood  part  of  the  vascular  bundles.     The 
cambium  shows,  in  the  longitudinal  view,  narrow,  elongated 
cells  with  end  surfaces  more  or  less  inclined,  and  touching 
each  other,  out  of  which  the  Avood  and  bast  proceed,  and 
low  broader  cells  which  continue  into  the 
'0  <^^  »^       ■hi      medullary  rays  on  both  sides. 
r  '     *""^^^  .  f^^^^  In  order  to  examine  the  sieve-plates 

^ii^'^^^^-'.n  ■      ^^i       (5),    we  make  a   radial   section   again 


Irom  the  alcohol  material,  and  lay  it  in 
an  aqueous  solution  of  aniline  blue  (6). 
eIIj  "^^  bij^rtj  After  a  few  miiuites,  transfer  it  to  glyc- 
pl/^|l'  11  if  ei'iiie  on  the  slide.  The  glycerine  takes 
Fi*^'^.  I'l  Pi  the  coloring  matter  from  all  of  the  tissue 
I '  "]  ;  5-  ^  ||?  except  the  sieves,  and  makes  it  impos- 
k^  ^  JjJ  v^  sible  to  overlook  them.  The  color  is  a 
beautiful,  durable  blue,  and  the  prepara- 

FiG.    48.     rinus    syl-     .-  i  j  t         -\tit 

vestris.  Parts  of  two  ^'ou  may  be  made  permanent.  We  can 
sieve-tubes  with  sieve  distinguish  the  sievc-plates  in  the  near 
vicinity  of  the  cambium,  and  follow  out 
the  same  into  the  region  where  the  sieve-tubes  are  crushed, 
and  the  sieve-plates  lose  thereby  their  radial  position. 
Still,  before  that,  the  sieve-plates  have  lost  their stainable 
quality.  The  sieve-tubes  have  the  form  of  cambium  cells 
and  have  the  sieve-plates  on  their  radial  walls,  as  the  tra- 
cheids have  the  bordered  pits.  The  sieve-plates  are,  for 
the  most  part,  smaller  than  the  bordered  pits  ;  they  appear 
as  round  or  oval  spots,  which  are  divided  info  an  indeünite 
number  of  finely-dotted  fields  with  angular  outHne,  Fig.  48. 


STRUCTURE    OF   CONIFEROUS    STEMS.  115 

At  some  distance  from  the  cambium,  the  sieve-plates  are 
covered  with  the  callus-plate,  a  brilliant  blue  substance. 
Farther  away  these  disappear,  the  sieve-plate  is  naked  and 
colorless.  The  sieve-tubes  are  out  of  function  here.  It  is 
not  difficult  to  see  that  the  active  sieve-tubes  contain  pro- 
toplasm ;  still,  the  nucleus  is  wanting,  most  surprisingly, 
and  has  disappeared  even  from  the  youngest  sieve-tube. 
The  crystal-bearing  cells  of  the  bast  are  recognized  by 
their  brown  contents,  are  relatively  short,  meet  principally 
with  transverse  walls,  and  probably  are  produced  by  the 
transverse  division  of  the  cambium  cells.  They  have  im- 
merous  prismatic  crystals  lying  near  and  over  each  other. 
Beyond  these  are  the  starch-bearing  cells.  They  are  shorter 
than  the  crystal  cells,  lie  in  threads  over  each  other,  and 
are  intercalated  singly  or  in  a  long  series  between  the 
crystal-bearing  cells.  They  afterwards  swell  very  consid- 
erably. The  medullary  rays  may  be  easily  followed  from 
the  wood  through  the  bast.  They  retain  their  essential 
structure,  only  losing  the  characteristic  pitting.  The 
starch-bearing  series  are  always  inclosed  by  a  row  of  empty 
cells  above  and  below. 

The  resin-duct  in  the  longitudinal  section  appears  as  a 
long,  continuous  tube,  inclosed  by  shorter  cells  with  trans- 
verse division  walls  arching  more  or  less  into  the  duct. 
Sometimes  a  resin  passage  is  found  in  a  medullary  ray. 
Naturally,  it  follows  a  radial  course  and  passes  the  cambium 
from  the  wood  to  the  bast. 

Now  make  a  tangential  section  from  the  alcohol  mate- 
rial. It  should  be  made  both  in  the  bast  and  in  the  wood. 
The  wood  section  shows  the  tracheids  and  the  severed 
medullary  ray.  The  latter  have  a  spindle-shaped  outline 
Avhile  the  cells  towards  the  ends  become  smaller.  The 
narrowest  medullary  rays  have  three  cells,  the  majority 


116 


STRUCTURE    OF   CONIFEROUS    STEMS. 


eight ;  but  some  bave  as  many  as  twenty.  The  narowest 
are  one  layer  of  cells  in  thickness,  the  others  may  bave  sev- 
eral layers  in  the  middle,  and  in  that  case  may  have  a 
resin  passage  in  the  centre,  which  will  of  course  be  cut 
across.  Make  a  bast  section  by  cutting  away  from  the 
outside  a  number  of  sections  of  the  old  bast  till  at  last  we 
come  to  the  young  wood.  Examining  this  section  with  a 
low  power  we  inquire  first  what  the  still  active  «ieve-tubes 
contain.  We  look  for  the  callus-plates,  and  we  easily  see 
\  them,  without  staining  or  high  magni- 

fication, refractive  pads  lying  on  the 
cell  walls.  Treat  the  sieve-plate  with 
chloriodide  of  zinc  to  which  a  like 
quantity  of  potassic  iodide  of  iodine  di- 
luted with  half  water  is  added.  The 
image  of  the  sieve-plates  is  in  this  view 
much  the  same  as  in  cross-sections,  still 
the  number  of  them  is  much  greater  and 
il!  '  -"i'V  j<7(i  ^"  *^'^^  ^^  more  likely  to  hit  upon  one 
i"l  IK  I  '  i,'J  favorably  situated.  AVe  shall  find  what 
we  are  looking  for  soonest  in  the  edges 
of  the  section.  The  sieve-plate,  Fig.  49, 
A,  presents  itself  in  profile  within  the 

^,  before  the  foiniation  t    i  n  /•     ^i  •  i     i  mi 

of  ti,e  sieve  i.iate;  B,  I'-^^li'il  ^alls  of  the  sievc-tubc.  The 
after  the  same;  c,  sieve  walls  themsclves  are  souicwhat  swollen 

tube  whicli  has  passed  ii-i  ii-ti 

beyoudihcactivestage.  and  colored  violct  by  the  chloriodide 
X^^^-  of  zinc.      The  sieve-plate  is  stained  a 

red-broAvn  if  it  belongs  to  a  still  active  sieve-tube.  This 
staining  comes  from  the  presence  of  plasma  strings  which 
penetrate  both  sides  of  the  sieve  region.  The  sieve-plates 
look  as  though  they  were  traced  over  with  red-brown  cray- 
ons (see  the  figui-e).  The  callus-plate,  JJ,  in  case  the 
zinc  solution  is  not  strong  enough  to  dissolve  it,  is  stained 


Fig.  49.  Pinus  sylves- 
tris. Walls  of  sieve- 
<ubes  after  treatment 
with  cloriodide  of  zinc. 


STRUCTURE    OF   CONIFEROUS    STEMS.  117 

a  red-brown.  The  sieve-plates  of  those  tubes  which  have 
passed  their  active  stage  appear  a  clear  violet,  (J.  The 
plasma  strings  and  the  callus-plates  have  both  disappeared. 
Stain  the  section  with  aniline  blue  and  examine  in  gly- 
cerine and  the  luminous  blue  callus-plates  are  very  clearly 
seen.  We  can  easily  follow  the  grpwth  of  them  on  one 
side,  and  their  disappearance  on  the  other. 

Notes. 

(1)  Russow,  Bot.  Centralbl,  1883,  Bd.  xni,  p.  140. 

(2)  Sanlo,  Jahrb.  f.  wiss,  Bot.  Bd.  ix,  p.  51;  E.  Strasburger,  Zell- 
häute, p.  39. 

(3)  Nach  N.  J.  C.  Müller,  Jahrb.  f.  wiss.  Bot.,  Bd.  v,  p,  398. 

(4)  See  de  Bary,  Vergl.  Anat.  p.  505. 

(5)  Janczewski,  Mein,  de  la  Soc.  d.  Sc.  nat.  de  Cherbourg.  Vol. 
XXIII,  p.  260 ;  E.  Strasburger,  Zellhäute,  p.  57 ;  Russow,  Dorp.  naturf. 
Gesellsch.,  17  Feb.  1882,  p.  264. 

(6)  K.  Wilhelm,  Beiträge  zur  Kentniss  des  Siebröhrenapparates, 
1880,  p.  36;  Russow,  Stzber.  d.  Dorp.  naturf.  Gesellsch.,  1881,  p.  63. 


LESSON  XI. 

Structure  of  Linden.     Bicollateral  Vascular  Bun- 
dles OF  THE  Cucurbita.    Sieve-tubes. 

A  transverse  section  of  a  branch  of  the  linden,  Tilia 
parvifoUa,  5  mm.  thick,  shows  us  a  large-celled  pith  whose 
air-filled  cells  are  grouped  rosette-like  about  single  nar- 
rower cells  filled  with  fine-grained  brown  contents.  In 
the  outer  parts  of  the  pith  are  gum  reservoirs  which,  be- 
ing as  yet  empty,  form  cavities  in  the  parenchymatous 
tissue.  On  the  outermost  edges  the  pith  is  small-celled 
and  the  cells  filled  with  finely  granular  contents.  Into  this 
small-celled  tissue  projects  the  primary  wood  part  of  the 
vascular  bundle.  The  spiral  vessels  are  seen  in  the  section 
by  their  occasional  prominent  thickened  band.  We  count 
some  five  annual  rings  in  a  transverse  section  of  5  mm.  in 
diameter.  The  spring  growth  produces  large  vessels  close 
together  which  distinctly  mark  the  boundary  of  the  year. 
Beyond,  the  wide  vessels  stand  singly  or  in  single  groups, 
and  in  the  last  phases  of  vegetation  the  cambium  forms 
only  narrow  cells.  Beyond  the  cambium,  the  most  con- 
spicuous object  is  the  wedge-shaped,  pointed  bast.  In  this 
is  a  tangentially-arranged  lighter  and  darker  stripe.  The 
light  stripe  is  composed  of  numerous  bast  fibres  solidly 
united  together,  whose  walls  are  so  thickened  as  to  reduce 
the  cell  cavity  to  a  dark  point.  The  stripes  have  an  irreg- 
ular contour ;  may  even  be  broken  apart.  The  darker 
stripes,  consisting  of  narrow,  starch-bearing  cells,  are  bast 
parenchyma  and  principally  rest  on  the  bast  fibres.  Near 
the  middle  of  the  layer  are  wide  cells,  among  which  we 
(118) 


STRUCTURE    OF   LINDEX    WOOD.  119 

recognize  sieve-tubes.  Small  cells,  apparently  cut  off  from 
the  corners  of  these,  are  the  conducting-cells.  There  are 
about  twice  as  many  stripes  of  secondary  bast  fibres  as 
there  are  yearly  rings  in  the  wood.  Excepting  the  two 
first  years,  two  bast  fibre  layers  are  regularly  produced 
each  year.  The  outer  edge  of  the  figure  will  have  been 
taken  from  the  primary  bast  string,  which  differs  in  no 
way  from  the  secondary  bast  string.  The  primary  medul- 
lary rays  are  mostly  two,  but  sometimes  more  cell-layers 
thick,  the  secondary  of  but  one.  They  may  be  traced 
through  the  cambium  to  the  primary  rind  or  into  the  bast. 
The  primary  rays  are  considerably  extended  and  separate 
the  bast  parts.  The  numerous  tangential  divisions  in 
these  medullary  rays  cause  the  cells  to  be  tangentially  ar- 
ranged. The  outer  end  of  the  medullary  rays  and  the 
primary  parts  of  the  bast  disappear  in  the  living  green 
primary  rind.  In  the  latter  and  in  the  extreme  ends  of 
the  rays  are  numerous  clusters  of  crystal.  Beyond  the 
chlorophyll-containing  cells,  are  the  collenchyma  cells  with 
their  white,  thickened  corners.  The  surface  of  the  stem 
is  covered  with  a  regularly  developed  periderm  whose  flat 
cells  are  the  measure  of  their  age,  that  is,  they  grow  more 
and  more  broAvn  toward  the  outside. 

The  radial  section  shows  that  the  vessels  of  the  second- 
ary wood  are  border-pitted  and  that  between  the  pits  is  a 
spiral  band,  or  inner  thickened  layer.  The  vessels  are 
joined  at  the  ends  by  inclined  walls  with  one  large  open- 
ing. Besides  the  vessels,  especially  in  the  autumn  wood, 
and  connected  with  them  by  transitional  forms,  are  the  tra- 
cheids,  thickened  like  the  vessels  and  with  both  ends 
pointed  and  closed.  Between  the  vessels  and  the  tracheids 
are  stretched  out  the  Avood  fibres  with  small,  infrequent, 
bordered  pits  and  narrow  wood-parenchyma  cells,  filled 
with  oil  drops  or  starch,  the  longitudinal  and  transverse 
walls  of  which  are  furnished  with  unbordered  pits.     The 


120  STRUCTURE    OP   LINDEN   WOOD. 

wood  fibres  are  longer  than  the  tracheids,  have  no  living 
contents  and  bear  only  water.  The  pits  of  the  wood  fibres 
open  into  the  cell  cavity  with  a  narrow  slit  which  is  in- 
clined in  a  direction  opposite  to  that  of  the  corresponding 
pit  in  the  adjoining  cell.  So  with  a  medinm  adjustment 
of  focus  a  small  cross  is  seen  in  the  pit.  In  these  wood 
fibres,  as  almost  universally  in  the  mechanical  elements 
(the  stereids),  the  stoma-like  pits  rise  toward  the  left, 
that  is,  they  follow  a  left  hand  spiral  line  (1).  Only 
where  one  vessel  borders  upon  another  or  upon  a  tracheid 
are  the  pits  in  the  walls  large  or  numerously  developed. 
Those  of  the  walls  bordering  on  the  wood  fibres  are  like 
them  sparsely  pitted.  The  pits  of  the  vessels  where  they 
touch  the  walls  of  the  wood  parenchyma  are  bordered 
only  on  one  side.  The  autumn-grown  wood  fibres  are  very 
narrow.  The  medullary  rays  appear  as  cross  stripes  of 
considerable  height  in  the  wood.  They  consist  of  right- 
angular,  radially-elongated  cells,  bearing  starch,  and  par- 
ticularly in  the  tangential  walls  very  numerously  pitted. 
In  the  bast  are  seen  the  very  long,  thick,  white,  bast 
fibres;  between  the  strings  of  bast  fil)res  short  parenchyma 
cells  provided  with  transverse  walls  and  bearing  starch 
and  sometimes  crystals,  and  the  sieve-tubes,  whose  sieve- 
plates  when  diagonally  placed  appear  to  be  distributed  into 
several  sections  by  cross  bands.  Outside  of  this,  the 
collenchyma  and  the  cork  öfter  something  of  interest. 
But  since  these  cells  are  of  equal  height  and  breadth  they 
present  the  same  image  in  this  as  in  the  transverse  section. 

The  tangential  section  confirms  the  conclusion,  drawn 
from  the  radial  section,  of  the  very  consideral)le  height  of 
the  medullary  rays.  These  rays  are  either  composed  of  a 
singlelayer  throughout  their  whole  height  or  are  double  in 
the  middle.  For  the  rest  we  find  the  same  elements  as  in 
the  radial  section. 

Turning  back  now  to  the  transverse  section,  we  shall  now 


STRUCTURE    OF   LINDEN   STEM.  121 

be  able  to  recognize  in  this  the  structure  of  the  wood. 
The  principal  mass  of  the  wood  is  formed  of  wood  fibres, 
and  in  tlie  autumn  growth  they  are  flatter  and  occur  al- 
most alone.  The  pits  of  the  fibres  are  difiicult  to  see,  and 
show  but  a  small  border.  The  vessels  and  tracheids  are 
clearly  border-pitted,  but  the  pits  are  very  numerous  only 
where  these  elements  touch  upon  each  other.  A  sharp 
distinction  is  with  difficulty  made  between  the  vessels 
and  the  tracheids  in  the  cross-section.  The  wood-paren- 
chyma cells  are  distinguished  by  their  smaller  width. 
They  lie  chiefly  about  the  vessels,  and  also  distributed 
singly  between  the  other  elements.  Their  starch  contents 
can  be  used  for  their  recognition  only  in  thicker  parts  of 
the  section,  because  in  the  thinner  places  the  starch  is 
scattered  over  the  cells  by  the  knife. 

Chloriodide  of  zinc  colors  the  wood  part  yellow-brown  ; 
the  cambium,  violet.  In  the  bast  there  is  a  beautiful  alter- 
nation between  the  violet  thin-walled  parts  and  the  clear 
yellow  thick-walled  bast  fibres.  The  elongated  medul- 
lary rays  and  the  primary  rind  are  violet,  the  cork  red- 
brown. 

Coralline  colors  the  wood  cherrj^-i-ed  ;  the  bast-fil)res,  a 
strikingly  beautiful,  brilliant  rose-red.  The  sieve-plates 
in  the  transection  appear  a  ibxy-red. 

On  account  of  the  difficulty  of  studying  the  secondary 
wood  we  will  proceed  to  separate  the  elements  by  macer- 
ation and  examine  them  separately.  We  will  use  Tilia 
jyarvi/oUa  and  proceed  as  with  AristolocJda  and  disin- 
tegrate the  macerated  section  with  the  needles,  finding  the 
princii)al  pnrt  of  the  wood  to  consist  of  fibres.  Fig.  50, 
A,  B.  The  swelling  of  the  walls  has  much  diminished 
the  size  of  the  obliquely-arranged,  slit-like  pits.  The 
short  parenchyma  cells  are  recognized  by  their  contents, 
either  separated  or  still  joined  together,  G,  and  resembling 


122 


ELEMENTS    OF    LINDEN   WOOD. 


in  their  outline  the  wood  fibres,  among  which  they 
lie  scattered  about.  The  tracheids  with  spiral  bands 
are  less  numerous  and,  in  contour  some,  E,  resem- 
ble the  wood  fibres,  others,  Z),  the  vessels.  Finally, 
we  C(mie  to  the  vessels  separated  into  sections  as  F^ 
or  formins:  long  tubes.      There  are  Ions:  bast  fibres 


V 

c 


Fig.  so.  Tilia  parvifolia.  Cells  from  the  secondary  wood  and  bast  iso- 
lated by  maceration.  A  and  B,  wood  fibres;  C,  wood  parenchyma;  D 
and  E,  tracheids;  F,  vessels;  G,  bast  fibres.  X  ISO. 


with  very  narrow  cell  cavities,  G.  An  attentive  ex- 
amination of  the  tracheids  and  the  vessels  demon- 
strates that  the  slit-like  orifices  of  the  pits  have  an 
opposite  inclination  to  the  spiral  bands,  in  the  ves- 
sels being  much  steeper  and  in  the  narrow  tracheids 


VASCULAR   BUNDLES    OF   CUCURBITA.  123 

about  as  steep  as  they.  The  tracheids  and  the  vessels  being 
so  much  alike  it  is  often  difficult  to  distinouish  a  wide  tra- 
cheid  from  a  narrow  vessel,  unless  indeed  the  end  be  per- 
forated and  it  is  not  always  easy  to  determine  this  point. 
But,  in  fact,  this  distinction  is  of  no  great  account  since  the 
two  elements  pass  into  each  other  by  minute  gradations. 
So  we  classify  them  by  their  forms  and  call  the  tube-like 
forms  vessels  and  the  fibre-like  forms  tracheids. 

Taking  now  the  Cucurbita  pepo  for  examination,  we 
find  that  the  vascular  bundle  has  two  bast  parts,  one  on 
the  inside  and  one  on  the  outside  of  the  wood.  These 
bundles  are,  therefore,  bicollaterally  built;  the  outer  bast 
is  separated  from  the  wood  by  the  cambium  ;  the  inner  im- 
mediately joins  the  wood.  To  find  a  full-grown,  vascular 
bundle,  take  a  section  of  the  stem  from  a  point  half  a 
metre  from  the  end,  where  it  is  some  8  mm.  thick.  At  a 
point  somewhat  nearer  the  growing  end,  where  the  stem 
has  a  diameter  of  5  to  6  mm.,  the  larger  vessels  are  not 
yet  fully  developed.  Use  alcohol  material  for  the  inves- 
tigation. The  vascular  bundle  has  m)  sheath  and  is  not 
very  sharply  separated  from  the  surrounding  fundamental 
tissue.  The  image  will  be  improved  by  appljnng  aniline 
blue  to  the  section  for  a  short  time  and  then  examinino- 
it  in  glycerine.  The  portion  to  which  the  vascular  bundle 
belongs  will  take  a  darker  stain  than  the  fundamental  tis- 
sue. Excluding  for  the  moment  the  iimer  sieve  part,  Ave 
find  the  remainder  of  the  vascular  bundle  to  be  so  like  those 
alread}''  known  in  the  Ranunculus  and  the  Ghelidonia  that 
w^e  should  without  difficult}^  class  it  in  the  same  group. 
Observing  first  the  transverse  section  of  a  fully-developed, 
vascular  bundle,  with  complete  vessels,  we  look  for  the 
most  normal  case  where  there  are  two  large  vessels  ;  these 
are  among  the  widest  vessels  known.  Between  them  lie 
primary,  wood-parenchyma  cells,  pretty    Avide,  radially 


124 


VASCULAR   BUNDLES    OF   CUCURBITA. 


elongated  for  the  most  part  and  walls  with  net-like  thick- 
enings. 

Towards  the  inside  are  vessels  of  considerably  less  di- 
ameter than  the  two  described,  and  farther  on  are  others 
still  smaller.  Between  these  are  thin-walled  wood  paren- 
chyma which  continue  beyond  the  bounds  of  the  inmost 
vessels.     The  inner  bast  joins  these  and  is  composed  of 


Fig.  51.  Cucurbita  2762)0.  Parts  of  sieve-tubes.  .4,  transection;  i?  to  Z>,  longi- 
tudinal section;  A,  a  sieve-plate  from  above;  B  and  C  the  adjoining  parts  of  two 
sieve-tubes  from  the  side;  D,  the  connecting  part  of  the  strings  of  mucilage  from 
two  sieve-tubes  after  treatment  with  sulphuric  acid;  s,  conducting  cells;  u,  muci- 
lage string;  ;;r,  protoplasmic  utricle;  c,  callus-plate;  c*,  small  lateral  callus  plate 
of  a  lateral  sieve  spot.  X  510. 


wide  sieve-tubes,  narrow  conducting-cells  and  bast-paren- 
chyma. The  transversely-placed  sieve-plate  may  be  eas- 
ily seen  from  above,  Fig.  51,  A.  The  conducting-cells, 
A,  s,  show  very  plainly  with  their  contents  tinged  a  dark 
blue.  On  the  outer  side  of  the  wood  the  thin-walled,  radi- 
ally-arranged, cambium  cells  follow  directly  upon  the  two 
large   vessels  and  the  wood-parenchyma  lying  between. 


VASCULAR   BUNDLES    OF   CUCURBITA.  125 

Then  comes  the  outer  bast  which  is  constructed  like  the 
inner.  The  sieve-phites  are  easily  found  in  both  bast 
regions,  and,  according  to  our  magnification  appear  to  be 
perforated  with  small  or  large  pores.  In  the  older  sieve- 
tubes,  the  pores  are  narrower  and  divested  of  strongly- 
refractive  substances.  So  in  A,  Fig.  51,  the  sieve-plates 
are  often  covered  with  masses  of  violet-blue  matter. 
In  the  narrower  sieve-tubes  on  the  outer  and  inner  edges 
of  the  vascular  bundles  appear  the  callus-plates  as  homo- 
geneous, azure-blue  masses.  Focussing  deep  enough,  we 
strike  the  meshwork  of  the  sieve-plate.  Using  a  low 
power  on  a  transection,  we  see  that  the  vascular  bundle 
stands  arranged  in  two  rings.  The  vascular  bundles  of 
the  outer  ring  stand  before  the  edge ;  those  of  the  inner 
ring  alternate  with  those  of  the  outer.  A  rinof  of  scle- 
renchyma  fibres,  whose  elements  are  of  much  darker  color 
than  the  large-celled,  fundamental  tissue,  protects  the 
inner  part.  Upon  these  follow,  towards  the  outside,  rind- 
parenchyma  containing  chlorophyll,  and  then  typically 
developed,  here  and  there  interrupted,  uncolored,  bril- 
liant-white collenchyma. 

At  the  points  of  interruption,  the  rind  parenchyma 
reaches  through  to  the  epidermis,  which  latter  bears  the 
stomata  at  these  places.  The  stem  is  hollow  on  the  in- 
side. Cross-sections  of  the  stem,  where  it  is  not  more 
than  5  or  6  mm.  thick,  show  the  large  vessels  and  the 
cells  lying  between  in  the  state  of  formation.  It  often 
happens  that  of  the  two  larger  vessels,  only  one  is  being 
formed,  the  other  (m  the  contrary  being  obliterated  ;  then 
the  one  attains  an  almost  colossal  diameter.  In  many  cases 
also,  both  vessels  are  obliterated.  Finally,  there  are  in- 
dividual instances  in  which  both  vessels  occur  and  both 
are  as  large  as  is  usual  where  there  is  only  one. 


126  VASCULAR    BUNDLES    OF    CUCURBITA. 

Radial  sections,  rightly  taken,  show  us  that  the  nar- 
rowest vessels  are  the  ring  and  spiral  vessels  :  the  widest, 
the  pitted  with  ring-like,  transversely-placed  diaphragms. 
The  two  largest  vessels  have  walls  with  irregular,  net-like 
thickenings  with  numerous  pits  between  the  meshes  of  the 
net.  Sometimes  these  vessels  will  be  found  with  entire 
partition  walls,  in  which  case  a  nucleus  and  a  thin  layer  of 
protoplasm  on  the  walls  will  be  found.  Many  partition 
walls  will  be  found  swollen  in  the  middle  in  the  form  of  a 
biconvex  lens.  Longitudinal  sections  of  the  next  older 
parts  of  the  stem  will  show  us  in  place  of  these  partition 
walls  small  rings  affixed  to  the  side  walls,  the  nucleus  and 
protoplasmic  layer  having  disappeared.  Between  the  nar- 
row vessels  is  thin-walled,  primary,  wood-parenchyma 
tissue.  The  cells  between  the  large  vessels  belong  to  the 
thick-walled,  primary,  wood-parenchyma  tissue  ;  they  are 
thickly  pitted,  even  on  the  partition  walls.  The  walls  of 
these  cells  which  join  the  vessels  perpendicularly  are  wavy  ; 
this  causes  them  to  modify  the  pits  of  the  vessels.  In 
these  wood-parenchyma  cells  are  nuclei  and  a  protoplasmic 
sac. 

At  both  sides  of  the  vascular  bundles  we  may  conven- 
iently study  the  wide  sieve-tubes  (2),  Fig.  51,  B.  Stain 
the  section  with  aniline  blue  and  examine  in  glycerine. 
The  latter  fluid  will  withdraw  the  color  somewhat  from 
the  cell  walls  after  a  little  while,  but  not  from  the  cell  con- 
tents. Most  of  the  sieve-plates  are  transversely  placed, 
few  inclined.  Most  of  them  also  are  covered  with  a  cal- 
lus substance,  and  are  correspondingly  thickened.  See  Fig. 
51,  C.  Use  a  comparatively  low  power.  The  sieve-plates 
are  colored  a  pure  blue.  In  the  tubes  which  show  the 
sieve-plates  is  a  sac-like  axillary  string,  u.  It  is  a  muci- 
lasrinous  cord  widened  at  the  end  so  as  to  cover  the  whole 


VASCULAR   BUNDLES    OF   CUCURBITA.  127 

of  the  sieve-plate,  and  colored  an  indigo  blue.  The  end 
setting  on  the  sieve-plate  is  more  thickly  filled  with  con- 
tents. See  in  B.  The  collection  of  cell  contents  is  to  be 
remarked  in  one  or  both  ends  of  the  sieve-tube;  and,  if 
in  but  one,  at  the  upper  end.  Besides  this,  a  layer  of  pro- 
toplasm may  be  found  on  the  walls  of  the  sieve-tubes,^)'. 
No  nucleus  exists.  In  somewhat  younger  sieve-tubes  the 
mucilaginous  cord  may  be  seen  by  low  magnification  press- 
ing through  the  pores  of  the  sieve-plate  into  the  adjoin- 
ing sieve-tube. 

In  each  plate  the  strands  are  all  moving  in  one  direction, 
but  in  successive  plates  they  may  be  going  in  opposite 
directions.  The  phenomenon  is  not  seen  in  older  sieve- 
tubes,  the  callus  substance  having  increased  on  the  sieve- 
plate  and  narrowed  the  sieve  portion,  and  through  these 
narrowed  pores  the  slimy  contents  of  the  cell  continually 
pass  (see  B)  from  one  to  the  other.  On  the  outer 
and  inner  edges  of  the  vascular  bundle  the  callus-plates 
cover  the  sieve-plates,  Fig.  51,  C.  They  are  colored 
azure  blue,  and  show  the  sieve-plate  in  their  midst  more 
or  less  clearly. 

The  callus-plates  consist  of  two  halves  which  belono- 
respectively  to  two  neighboring  sieve-tubes  and  are  con- 
nected through  the  pores  in  the  sieve-plates.  A  delicate 
striation  is  often  observable  which  extends  throuo-h  the 
connecting  pores.  See  the  Figure.  When  two  sieve- 
tubes  are  laterally  joined,  small  sieve-spots  appear  on  the 
common  wall  and  afterwards  a  one-sided,  c*,  or  two-sided 
callus-plate  occurs.  Condncting-cells,  s,  of  the  same 
length  as  the  sieve-tubes,  follow  their  course.  They  have 
rich  contents  and  a  nucleus.  Sieve-tubes,  in  the  process 
of  development,  show  indigo-blue  colored  drops  of  muci- 
lage in  their  protoplasmic  wall-layer.    For  comparison,  it 


128  VASCULAR   BUNDLES    OF    CUCURBITA. 

is  necessary  to  make  a  longitudinal  section  of  fresh  ma- 
terial. The  sio^e-plates  appear  as  plainly  as  in  the  alco- 
hol material.  The  slimy  collections  on  the  sieve-plates 
are  easily  seen  ;  but  the  mucilage  nowhere  shows  itself 
drawn  back  from  the  side  walls  of  the  sieve-tubes  in  the 
form  of  a  string.  This  appearance  is  due  to  the  influence 
of  the  alcohol. 

Notes. 

(1)  See  Scliweudener,  Das  mech.  Princip.,  p.  8. 

(2)  Fortius  compare  principally  de  Bary,  Vergl.  Anatom.,  p.  179; 
K.  Willielm,  Beiträge  znr  Kenntniss  des  Siebröliren-Apparates  dicoty- 
ler  Pflanzen ;  E.  \.  Janczewski,  Etudes  compar^es  siu*  les  tubes  cri- 
breux,  Mem.  de  la  soc.  nat.  des  sc.  nat.  de  Cherbourg,  T.  xxin:  Russow, 
Stzber.,  der  Dorp,  naturf.  Gesellsch.,  Jahrg.  1881,  u.  1882. 


LESSON  XII. 

Vascular  Bundles  of  the  Axile  Cylinder,  and  the 
Secondary  Lateral  Growth  of  the  Roots. 

For  the  study  of  the  axile,  vascular-bundle  cylinder 
of  the  roots,  we  w'ill  take  a  root  of  the  common  onion, 
Allium  cejM.     One  may  have  plenty  of  material  at  any 


Fig.  5'2.  Transection  from  the  base  of  a  large  ;idventive  root  of  AUmm  cepa. 
c,  rind;  e,  endoderm;  p,  pericambium;  fjf+Uj  I'^'S  vessel;  sp,  spiral  vessel;  sc  and 
sc,  scaliform  vessels ;  v,  bast.  X  240. 

time  by  putting  an  onion  in  a  glass  of  w^ater  and  letting  the 
roots  sprout.  Fig.  52  is  a  section  of  such  a  root  near 
the  base.  The  epidermis  and  the  very  stout  rind  tissue  are 
left  out  of  the  drawing.   Still  one  may  see  some  of  the  cells 

9  (129) 


130  VASCULAR   BUNDLES    OF    ONION    ROOT. 

bordering  the  endoderm  at  c.  The  endoderm,  e,  shows 
in  a  characteristic  manner,  dark  shadows  on  its  radial 
walls.  These  shadows  are  caused  by  the  wavy  bending 
of  the  middle  part  of  the  walls.  Such  an  endoderm  al- 
ways consists  of  one  layer.  We  have  already  met  it  in 
the  envelope  of  the  vascular  bundles  of  the  leaf  of  Iris, 
which  shows  it  to  be  not  confined  to  the  roots.  In  the 
middle  of  the  vascular-bundle  cylinder  there  are,  in  this 
case,  two  wide,  scaliform  vessels,  sc.  In  other  cases  more 
or  less  than  two  may  be  seen.  If  the  root  is  not  old 
enough,  the  central  and  perhaps  adjoining  vessels  will  be 
thin-walled,  not  fully  developed.  Adjoining  the  central 
vessel  or  vessels  are  almost  always  six  narrower  scaliform 
vessels,  sc*,  and  upon  the  latter  follows  a  group  of  quite 
narrow  spiral  andring  vessels,  sj),  S2)-\-ci.  The  size  of  the 
vessels  diminishes  from  the  centre  outward  and  the  outer- 
most are  the  ring  and  spiral  vessels.  Herewith  the  root 
diflers  from  the  stem.  The  wood  occupies  about  180°  of 
the  circumference  of  the  cylinder,  being  arranged  in  the 
form  of  a  six-rayed  star.  The  bast,  v,  alternates  with  the 
wood.  This  is  the  general  law  with  respect  to  the  axile, 
cylindrical,  vascular  bundle  of  the  roots.  A  layer  of 
parenchymatous  fundamental  tissue  laterally  separates  the 
wood  from  the  bast.  The  latter  is  recognized  b}^  the 
M'hite  sparkling  walls  of  its  cells.  It  consists  of  sieve- 
tubes  and  conductino'-cells  not  distinguishable  with  cer- 
tainty  in  a  cross-section.  The  vessels  and  the  bast  are 
separated  from  the  endoderm  by  a  single  layer  of  peri- 
cambium  cells.  Concentrated  sulphuric  acid  will  dissolve 
the  whole  section  with  the  exception  of  the  epidermis  and 
the  cell-layer  bordering  upon  it,  and  the  vessels  and  the 
endoderm.  The  latter  will  be  colored  a  beautiful  yellow. 
The  endoderm,  which  will  indeed  in  part  bend  about  dur- 
ing the  action  of  the  acid,  shows  the  middle  band  in  its  ra- 


VASCULAR  BUNDLE  OF  FLAG  EOOT. 


131 


dial  walls  beautifully  uiKlulatecl.  A  similar  appearance  is 
observable  on  the  outer  layer  of  rind  cells  bordering  ou 
the  epidermis.  The  cells  in  question  are  bound  fast  to- 
gether under  each  other  and  form  a  sort  of  outer  endoderm 
which  is  sometimes  known  as  the  epidermoidal  layer(2). 
A  longitudinal  section  shows  us  the  vessels  already  men- 
tioned ;  and  staining  with  coralline,  the  sieve-tubes  and 
sieve-plates  colored  rose-red  are  easily  made  visible.  The 
conducting-cells,  shorter  and  tilled  with  contents,  are  easily 
distingnished.  The 
undulation  of  the  mid- 
dle band  of  the  radial 
Avails  of  the  endoderm 
cells,  looked  at  from 
the  surface,  appear 
like  a  scab  form  thick- 
ening. The  pericam- 
bium  cells  have  the 
same  form  as  the  en- 
doderm cells,  yet  of 
greater  length.  The 
inner  endoderm  takes 
the  color  (coralline) 
with  some  avidity, 
while  the  outer  endoderm  is  contrasted  from  the  surround- 
ing tissue  by  its  coloilessness. 

We  will  take  for  further  study  a  section  of  the  root  of 
Aco7'us  calamus,  sweet  Hag,  shown  in  Fig.  53.  The  vas- 
cular rays  do  not  meet  in  the  centre  of  the  cylinder,  but 
are  arranged  in  eijjht  seö-nients  of  a  circle,  while  the  mid- 

BOO  ' 

die  is  tilled  with  medullary  tissue.  The  larger  vessels  lie 
nearest  the  centre,  the  smaller  toward  the  periphery.  The 
bast,  V,  alternates  with  the  vascular  rays.  The  two  are 
laterally  separated  by  a  single  or  double  layer  of  paren- 


FiG.  53.  Transection  through  the  root  of  Aco- 
rus calnmus.  m,  pilh;  s,  wood;  v.  bast;p,  peri- 
cauibiuai ;  e,  endoderm ;  c,  rind.   X  'j^- 


132  VASCULAR   BUNDLE    OF    FLAG    ROOT. 

cbymatous  fundamental  tissue,  and  from  the  endoderm, 
e,  ])y  a  single  layer  of  pericamhium.  The  endoderm  con- 
sists of  fiat  thin-walled  cells,  and  it  and  the  pericambium 
and  all  the  remaining  fundamental  tissues  of  the  vascular- 
bundle  cylinder  are  filled  with  starch,  which  makes  the 
starchless  bast  appear  verj''  distinct  in  the  image.  The 
cells  of  the  inner  rind  are  separated  into  single-celled 
layers  by  numerous  air  passages.  In  the  periphery  the 
rind  cells  are  compacted  together  into  a  solid  layer,  several 
cells  thick.  The  outer  hypodermal  rind  layer  consists  of 
radially-elongated  cells  and  forms  in  this,  as  in  other  roots, 
an  outer  endoderm  which  persists,  while  the  epidermis 
dies  and  disintegrates.  Add  potash  lye,  and  dissolve  the 
starch,  and  the  dark  shadows  in  the  radial  walls  of  the 
endoderm  are  distinctly  seen.  Treat  with  sulphuric  acid, 
and  we  see  that  the  whole  cell-wall  of  the  outer  endoderm 
is  cuticularized,  but  only  the  shadow-forming  band  of  the 
inner  endoderm.  The  cells  of  the  outer  endoderm  con- 
tain resin.  There  is  a  mechanical  significance  to  these  en- 
doderms.  They  protect  both  the  surface  and  the  axile 
vascular-bundle  cylinder.  By  the  suberization  of  their 
cell-walls  they  attain  great  solidity  and  little  extensibility. 
But  the  interpassage  of  fluids  between  the  vascular-bundle 
cylinder  and  the  rind  is  not  thereby  interfered  with,  since 
the  cells  of  the  inner  endoderm  are  suberized  only  or  prin- 
cipally on  their  radial  walls  (3). 

A  cross-section  of  a  root  of  Iris  florentina  shows  us  an 
axile  vascular-bundle  cylinder,  quite  exactly  like  that  of 
the  Acorus,  except  that  the  endoderm  is  differently  built. 
See  Fig.  54.  The  cells  are  unilaterally  thickened,  U-shaped 
and  the  thickening  beautifully  laminated,  e.  Exactly  in 
front  of  the  vascular  ray  is  a  single  unthickened  cell,  f. 
It  is  known  as  a  transit-cell  (4),  and  being  permeable 
maintains  the  connection  with  the   surrounding    rind,  c. 


VASCULAR    BUNDLE    OF    IRIS    ROOT. 


133 


The  thickened  laj'er  swells  and  dissolves  in  concentrated 
sulphuric  acid,  the  cuticularized  middle  lamella  only  re- 
maining and  forming  a  delicate  envelope  about  the  endo- 
derm and  transit  cells.  The  middle  lamelhi  between  the 
vessels  and  in  the  pith  is  not  dissolved,  but  forms  a  delicate, 
yellowish-brown  network.  A  tangential  section,  which  just 
grazes  the  endoderm,  teaches  us  that  the  longitudinal  stripe, 
which  lies  in  front  of  the  wood  parts,  consists  of  long, 
thickened,  alternating  Avith  short,  unthickened,  transit  cells, 
full  of  cell  contents.  Some- 
times, two  short  transit  cells 
follow  each  other. 

The  roots  of  dicotyledons 
are  less  favorable  for  study 
than  those  of  the  monocoty- 
ledons, but,  having  become 
acquainted  with  the  latter, 
we  shall  have  no  difficulty 
with  the  former.  Make  a 
cross-section  from  the  base 
of  an  adventive  root  of  a  run- 
ner of  Ranimculus  rej)enS.  fig.  54.  Part  of  a  transection  thronüli 
m\  -1  11  II        the  root  of  Iris  ßoreniina.   e,  endoilerm ; 

The       axile      vascular-bundle    ;,,  pericambium; /transit  cell;  .-.bast; 

cylinder  is  not  so  sharply  «,  vessels  in  the  wood;  c,  rind,  x  210. 
diflerentiated  from  the  rind  tissue  as  in  the  monocotyledons, 
but  by  attentive  examination  we  shall  find  on  the  border 
of  the  two  the  dark  shadows  which  mark  the  endoderm. 
The  axile  cylinder  is  divided  into  four  or  five  vascular  rays, 
according  to  the  size  of  the  root.  The  laro'er  vessels  lie  here 
towards  the  inside  and  the  smaller  towards  the  outside.  In 
monocotyledons,  the  innermost  vessels  are  distinguished 
by  their  large  size.  This  is  rarely  seen  in  the  dicotyledons 
and  not  at  all  in  the  lianunculus.  The  vascular  rays  ex- 
tend to  the  middle  of  the  cylinder,  and  abut  more  or  less 


134  VASCULAR   BUNDLES    OF   JCJXIPER    ROOT. 

fiill_y  against  each  other.  The  innermost  vessels  are  latest 
in  developing,  and  remain  in  the  condition  of  thin-walled 
elongated  cells.  The  bast  alternates  in  the  ordinär}^  way 
with  the  wood. 

The  roots  of  the  vascnlar  cryptogams  are  simpler,  and 
3^et  are  constrncted  on  the  same  type  as  those  of  the  phan- 
erogams. 

Take  next  a  rootlet  of  Taxus  baccafa,  abont  1  mm.  thick, 
and  make  a  cross-section.  The  rind  consists  of  about  ten 
thicknesses  of  parenchyma  cells.  The  outer  cell  layer  of 
the  rind  is  not  especially  ditferentiated,  there  being  no  dis- 
tinct epidermis.  The  inside  of  the  section  is  filled  with  the 
axile  vascular-bundle  cylinder,  which  is  surrounded  by  an 
endoderm.  The  latter  consists  of  flat,  thin-walled,  sub- 
erized  cells,  whose  walls  are  browned,  and  wdiose  diameter 
is  considerabl}-^  less  than  that  of  the  rind  cells,  the  radial 
walls  being  characteristically  shaded.  A  single-celled, 
thickened  layer  is  developed  about  the  endoderm.  The 
cells  are  of  the  same  width  as  those  of  the  rest  of  the  rind, 
but  the  radial  walls  are  furnished  with  a  thick,  bright, 
yellow  ring.  These  ring-like  thickenings  correspond  to 
that  in  the  neighboring  cells,  which  give  them  in  section  the 
form  of  a  biconvex-lens.  The  axile  vascular-bundle  cylin- 
der shows  a  double-arched  wood  body,  extending  across 
it,  at  the  opposite  ends  of  which  is  a  narrow,  spiral  ves- 
sel. Inward,  and  joining  these,  is  a  strip  of  tracheids 
with  bordered  pits,  characteristic  of  the  conifers.  They 
are  easily  recognized  by  their  clear  yellow,  strongly-thick- 
ened walls.  These  tracheids  almost  always  meet  in  the 
middle  of  the  cylinder,  forming  a  plate.  On  each  side  of 
the  tracheids,  lies  a  strip  of  narrow,  thin-walled,  funda- 
mental-tissue cells,  bearing  starch.  Upon  these,  borders 
a  somewhat  small-celled  tissue  of  thin-walled  bast.  Fi- 
nally, beyond  this,  a  large-celled  starch-bearing  layer,  about 


AXILE    VASCULAR    BUNDLE    OF   JUNIPER.  135 

four  cells  thick.  The  latter  are  joined  together,  making 
a  complete  circle,  somewhat  reduced  before  the  spiral  ves- 
sels.    They  represent  the  pericambium. 

If  now  we  examine  a  cross-section  about  1.3  mm.  in 
diameter,  we  shall  find  the  two  sides  of  the  plate  of  tra- 
chekls  dividing  and  becoming  transformed  into  cambium, 
which  produces  tracheids  on  the  inside  and  bast  on  the 
outside,  and  on  both  sides  mednllary  rays.     Now  examine 


Fig.  55.  Transection  of  root  of  Taxus  baccata  after  the  beginning  of  the  lateral 
growth,  c,  rind ;  m,  tliickening  layer;  e,  enrtodenn;  jo,  pericambiuni ;  s,  spiral  ves- 
sels; t,  primary  trachcid  plate;/,  stripe  of  fundamental  tissue;  t",  secondary 
tracheids  with  medullary  rays;  v",  secondary  bast;  v',  compressed  priniai-y  bast; 
k,  cells  in  secondary  bast  with  crystals  in  the  walls;  r,  resiubearing  cells  in  peri- 
cambium.  X  -l-' 

the  further  activity  of  the  cambium  in  a  section  of  a  root 
2  mm.  wide,  as  shown  in  Fig.  55.  It  shows  the  already 
well-known  relations  :  the  rind,  c,  which  has  lost  its  hairs 
from  the  onter  layer  of  cells ;  the  outer  strengthening 
layer,  m;  the  endoderm,  e;  and  the  axile  cylinder.     The 


136  AXILE    VASCULAR    CYLINDER    OF    JUNIPER. 

outer  cell-layer  of  the  pericambium  bus  in  the  mean n bile 
begun  to  divide  and  bas  been  transformed  into  a  few  layers 
of  peritlerm.  Ou  botb  sides  of  tbe  tracbeid  plate  we  see 
tbe  inner  inactive  layer  of  fuudaniental  tissue,/,  the  so- 
called  connective  tissue  :  beyond,  tbe  newly-formed  radi- 
ally-arranged tracbeids,  /'",  witb  numerous  intercalated 
medullary  vays.  Tbese  relations  are  more  easily  seen  if  one 
adds  a  little  potash  lye  to  tbe  preparation.  Tbe  vessels, 
6;,  on  the  edges  of  tbe  middle  plate  come  out  distinct  and 
black. 

The  tracbeid  plate,  /',  as  well  as  tbe  tracbeids  formed 
by  the  cambium,  y^  is  cobn-cd  a  beautiful  yellow.  Tbe 
connective  tissue  remains  white.  The  secondary  wood 
layers  have  a  plano-convex  outline  Avbicb  runs  to  a  point 
at  tbe  edges  but  not  here  in  front  of  tbe  vessels.  On  the 
outer  side  of  the  wood  body  we  find  the  cambium  and  out- 
side of  this  the  secondary  bast,  v",  which  after  treatment 
Avitlitbe  potash  appears  white  but  in  which  single  cells  are 
black,  k,  having  crystals  of  oxalate  of  lime  in  tbeir  walls. 
The  primary  bast  forms  a  layer  of  compressed  cells  on  the 
outside  of  the  secondary.  Tbe  potasb  brings  out  tbe  peri- 
cambium more  distinctly  than  before,  also  the  resin-bear- 
ing cells  witb  tbeir  j^ellow-brown  contents.  The  cork  layer 
produced  from  the  pericam])ium  is  colored  a  yellow-green, 
tbe  thickening  ring  of  the  strengthening  layer  a  brigbt 
yellow.     Tbe  endoderm  is  compressed  by  tbe  cork  layer. 

Making  a  section  now  of  a  root  2  mm.  thick  wbicb  bas 
thrown  off  its  rind  and  shows  a  dark  brown  surface,  we 
tind  tbe  section  has  a  fully  closed  wood  part,  and  makes  an 
image  indistinguishable  from  that  of  a  branch  of  like  size, 
were  it  not  that  here  the  place  of  the  pith  is  occupied  by 
tbe  primary  tracbeid  plate. 

The  vessels  at  the  edges  of  this  plate  are  somewhat  dif- 
ficult to  make  out.     Tbe  plate  is  inclosed  hy  the  starch- 


AXILE    VASCULAR    CYLINDER    JUNIPER.  137 

bcarina'  connective  tissue  which  here  to  a  certain  extent 
displaces  the  medulhiry  crown  and  into  which  the  oldest 
medullar}'  rays  open.  The  two  wood  bodies  have  united 
in  front  of  the  vascular  groups  and  the  medullary  rays  at 
that  place  are  scarcely  noticeable  by  special  width.  The 
surface  receives  the  inclosing  cork  layer  produced  from 
the  outermost  pericambium  layer.  The  secondary  rind 
consists  of  secondary  bast  and  the  elongated  medullary 
rays.  That  which  represents  the  primary  rind  here  will 
consist  of  the  enlarged  and  numerically  increased  pericam- 
bium cells  closely  packed  with  starch. 

Longitudinal  sections  show  that  the  middle  tracheid 
plate  consists  of  the  same  elements  as  the  secondary  wood. 
We  find  the  spiral  vessels  on  the  edges  of  the  plate,  and 
observe  that  the  cells  of  the  endoderm  are  quite  short  while 
those  of  the  strengthening  layer  are  far  larger  and  are 
even  longer  than  the  adjoining  cells  of  the  rind.  Coralline 
stains  the  tracheids  a  beautiful  coral-red  and  brings  out 
the  sieve-plates  in  the  primary  and  secondary  bast.  The 
rings  of  the  strengthening  layer  also  absorb  the  coralline. 

Notes. 

(1)  De  Bary,  Vergl.  Auat.,  p.  365,  where  the  older  literature  will  be 
fouud;  Olivier,  Ann.  d.  Sc.  nat.  Bot.,  vi  Ser.,  xi  Bd.,  p.  5,  Ö'. 

(2)  See  V,  Höhuel,  Stzber.  d.  k.  Ak.  d.  Wiss.  iu  Wien,  math,  uatur- 
wiss.  CI.  Bd.  Lxxvi,  i  Abth.  1877,  p.  642;  Olivier,  I.e. 

(3)  Schweudeuer,  Abh.  d.  kgl.  Ak.  d.  Wiss.  iu  Berlin,  1882.  The  pro- 
tective sheath  and  its  strengthening. 

(4)  See  last  work  quoted,  p.  13. 


LESSON  XIII. 
Vascular  Bundles  of  the  Ferns  and  Lycopods. 

In  the  leaves  and  stems  of  the  ferns  the  vascular  bnn- 
dles  are  concentrically  built,  whereby  the  wood  is  almost 
or  quite  fully  surrounded  by  the  bast. 

Make  a  section  of  Plens  aquilina,  in  which  it  is  possi- 
ble to  gat  a  good  knowledge  of  the  vascular  bundle,  even 
when  the  numerous,  sclerenchyma  strings  in  the  funda- 
mental tissue  do  not  permit  us  to  make  a  good  section. 
Make  the  section  from  the  rhizome  directly  behind  the 
growing  point  or  through  the  petiole  of  a  young  leaf. 
The  vascular  bundle  will  be  sufBcientl}'  developed,  while 
the  fundamental  tissue  will  not  be  much  hardened.  The 
bundle  will  be  the  same  in  the  rhizome  and  the  petiole, 
and  a  cross-section  of  it  from  the  base  of  the  latter  is 
shown  in  Fig.  56.  Choose  a  small  bundle.  We  first  no- 
tice the  large,  border-pitted,  scaliform  vessels,  sc;  still, 
the  smaller  vessels  are  thickened  also  and  only  the  few  on 
the  two  ends  of  the  wood  which  adjoin  the  protoxylem 
elements  have  spiral  thickenings,  sp.  The  vessels  are 
surrounded,  when  they  do  not  touch  each  other,  by  starch- 
bearing,  wood-parenchyma  cells,  Ip.  Wood-parenchyma 
and  vessels  form  the  wood  part  which  is  almost  perfectly 
enclosed  by  the  bast.  The  latter  borders  on  the  wood 
parenchyma  with  sieve-tubes,  v,  which  are  succeeded  out- 
wardly by  narrow  conducting-cells,  s,  wiiich  are  filled 
with  protoplasm — not  starch,  as  iodine  will  show.  But 
single,  starch-bearing  cells  are  sparsely  distributed  through 
this  tissue. 

(138) 


VASCULAR   BUNDLES    OF   FERNS. 


139 


The  periphery  of  the  bast  takes  on  a  layer  of  still  nar- 
rower, thick-walled  protophloeni  elements.  The  bast  is 
surrounded  by  a  simple  layer  of  cells,  j-jjj,  filled  with 
starch,  which  in  its  i)Osition,  but  not  in  its  origin,  resem- 
bles pericambinm  and  may  be  called  periphloem.  Around 
this  preliminary  sheath  is  the  endoderm,  e,  thin-walled, 
free    from   starch  and    suberized,  and  showinir  the  dark 


Fig.  5(i.  Transection  of  a  vascular  bundle  from  the  petiole  of  Pteris  aquUina. 
sc,  .^calilbi-m  vessels;  sp.  sjjiral  vessels.  In  sc*  a  piece  of  tlie  scaliform  perfoi'ated 
wall  is  seen;  //>,  wood  paroncliyma;  v,  sieve  tubes;  s,  conducting-cells;pr,  proto- 
phluL'm;^^;,  periphloem;  e,  endoderm.  X  2l0. 

shadows  on  the  radial  w^alls.  The  periphloem  and  en- 
doderm cells  correspond  to  one  another  and  suggest  a 
common  origin  in  the  same  mother-cell.  The  wood  at 
its  two  edges,  together  with  the  covering  of  w^ood-p;iren- 
chyma,  borders  directly  on  the  periphloem  or  on  the  proto- 


140  VASCULAR   BUNDLES    IN   FERNS. 

phloem.  At  these  two  points,  the  bast  is  either  wholly 
or  nearly  interrupted,  but  in  other  ferns  this  break  may 
not  occur.  The  walls  of  the  endoderm  cells  are  often 
broken  by  the  cutting  and  then  the  vascular  bundle  will 
be  separated  from  the  fundamental  tissue.  The  cells  of 
this  tissue,  bordering  on  the  endoderm,  are  sometimes 
much  thickened  and  then  are  colored  a  yellow-brown. 
The  cross-section  through  the  rhizome  shows  a  browned 
and  cuticularized  parenchymatous  tissue  under  the  deep 
brown  epidermis  which,  further  towards  the  inside,  is 
colorless  and  full  of  starch.  This  starch-bearing,  funda- 
mental tissue  is  penetrated  with  the  vascular  bundles  and 
the  red-brown  sclerenchyma  fibres ;  the  latter  form  plates 
which  run  between  the  vascular  bundles  more  or  less  par- 
allel to  them.  The  outer  bundles  are  in  immediate  con- 
tact with  the  endoderm  on  the  outside,  supported  by  such 
sclerenchyma  fibres,  which  here  represent  the  mechanical 
tissue.  In  the  inside  of  the  bast  the  relations  are  simi- 
lar, there  being  a  hypodermal  ring  of  red-brown  scleren- 
ch^'ma  fibres  which  rest  on  the  epidermis. 

A  longitudinal  section  shows  us  all  the  wide,  scali- 
form  vessels  again.  The  ends  are  much  inclined,  ladder- 
like, border-pitted  and  in  part  perforated  (1).  On  the 
side  walls  separating  the  two  vessels,  it  is  easy  to  see  that 
the  pits  are  bordered  on  both  sides,  and  the  closing  mem- 
brane has  a  thickened  torus;  but  on  the  walls  bordering 
on  the  wood-parenchyma  cells  the  pits  are  bordered  only 
on  one  side,  and  the  closing  membrane  has  no  torus. 
The  section  may  also  hit  one  or  the  other  of  the  spiral  ves- 
sels, and  the  plates  of  the  sieve-tubes  may  also  be  discov- 
ered, but  only  by  the  most  careful  examination  ;  the  latter 
may  be  found  much  more  easily  b}^  the  help  of  coralline 
staining,  which  also  will  shoAV  the  sieve-plates  much  in- 
clined and  parted  into  numerous  fields  by  thickened  bands  ; 
besides  these,  the  lateral  walls  of  the  sieve-tubes  bear  sieve- 


VASCULAR   BUNDLES    OF    FERNS.  141 

spots.  Together  with  the  sieve-tubes,  we  find  the  slender, 
condncting-cells  with  finely,  gi-anul.ir  contents  and  nucleus, 
and  in  contact  with  the  vessels  the  starch-bearing,  rela- 
tively-short, wood-parenchyma  cells.  Resembling  the  lat- 
ter, are  the  starch-bearing  cells  of  the  periphloem.  Small 
pores  are  seen  in  the  walls  of  the  long-pointed,  scleren- 
chyma  fibres  of  the  fundamental  tissue. 

It  will  be  interesting  to  make  a  transection  of  the  petiole 
of  Pohjpodium  vulgare.  The  vascular  bundles  are  very 
thickly  sheathed  about,  but  the  sheath  corresponds  not  to 
the  endoderm  but  to  a  strengthening  layer.  This  layer  is 
but  a  single  cell  thick  and  these  cells  are  thickened  only  (m 
the  inside  walls  and  are  there  colored  a  dark  brown.  The 
essential  endoderm  lies  immediately  within  the  strength- 
ening layer  and  is  scarcely  recognizable  on  account  of  its 
cells  being  flattened  down  by  the  pressure  of  this  layer. 
Next  within  comes  the  starch-bearing,  single  stratum  of 
periphloem  cells,  then  the  bast  tissue  consisting  of  cells, 
of  almost  the  same  width.  The  condncting-cells  are  dis- 
tinguished by  their  contents  and,  as  is  apparent,  are  mingled 
with  the  sieve-tubes.  The  closely  grouped  vessels  are 
surrounded  without  by  a  single  laj'er  of  starch-bearing 
wood-parenchyma  cells  which  at  the  two  small  edges  ot  the 
wood-part  may  extend  even  to  the  periphloem. 

Prepare  now  a  cross-section  of  the  petiole  of  Scolopen- 
drium  vulgare,  where  Ave  shall  find  the  two  vascular  bun- 
dles reduced  to  one.  Two  wood  portions  lie  apparently 
in  one  vascular  bundle ;  rather,  in  a  compound  bundle, 
either  near  each  other,  or,  as  is  frequently  to  be  seen, 
united  at  one  point  so  as  to  form  an  X-like  figure.  The 
stouter  legs  of  the  figure  are  turned  towards  the  upper  side 
of  the  petiole.  The  small  vessels  are  found  at  the  ends  of 
the  legs.  From  the  ends  of  the  upper  legs  small  vascular 
bundles  are  often  seen  branchins:  out.  The  cells  of  the  bast 
are  all  of  uniform  size,  but  the  condncting-cells  mingled 


142 


VASCULAR   BUNDLES    OF   FERNS. 


■with  the  sieve-tubes  are  easily  recognizable  l)y  their  con- 
tents. On  the  surface  of  the  tignre,  the  periphh^em  a})pears 
several  layers  thick  and  with  somewhat  thickened  walls. 
The  outer  circumference  of  the  compound  bundle  is  deeply 
fluted  at  three  points,  viz.,  above  and  at  the  two  sides  ;  and 
here  follows,  on  the  endoderm,  a  plate  of  sclerenchyma 
fibres,  red  brown,  and  thickened  almost  to  the  extinction 
of  their  cell  cavities.  Higher  up  in  the  leaf,  the  wood 
part   gradually  assumes    the    form    of  a  T.       The   three 

strenijthenino^  scle- 

«-/     renchyma     strings 

^     are  always  present 

even     though     re- 

duced. 

In  the  Lycoj)o~ 
dium  species  the 
axile  vascular-bun- 
dle cylinder  ap- 
pears in  a  relatively 
more  highly  com- 
plicated form.  Still 
the  relations  of  the 

Fig.  57.    Transection  of  stem  of  Lyc<podium  com-  partS      should      not 

planatum.    e/>,  epii lei  mis;  ve,  outer  s- heath  ;  H,  inner  ,        vi^       Uf  .  1- 

sheath;  pp,  periplilocm;   sc,  scallCoim   vessels ;  .sp,  DC  UlmCUll  10  nUUve 

ring  and  spiial  vessels ;  v,  sieve  elements.  X  2ö.  out    after    ll  a  V  i  n  o" 

seen  the  compound  vascular-bundle  in  the  petiole  of  the 
/Scolopendrium.  We  have  in  fact  to  deal  with  an  axile 
vascular-bundle  cylinder,  in  the  Lycopodium,  which  i« 
formed  by  the  blending  of  several  vascular  bundles  built 
like  that  of  the  last  example.  We  will  take  the  Lycopo- 
dium  cömjplanata,  but  another  species  will  do  as  well.  Color 
the  section  with  an  aqueous  solution  of  safranin.  Fig.  57 
represents  what  we  shall  see.  First  we  have  the  ei)ider- 
mis,  ep;  then  the  rind  cells  which  at  first  have  wide  cav- 
ities but  which  toward  the  inside  diminish  in  width,  and 


VASCULAR  BUNDLES  OF  LTCOPODIUM.       143 

increase  in  thickness  of  Wiill  till  they  form  an  almost 
solid  sclerenchyma  sheath  which  Ave  will  call  the  outer 
sheath,  ve.  Between  these  strongly  thickened  rind  ele- 
ments are  air-tilled,  intercellular  spaces.  The  outer  cells 
of  the  rind  are  colored  by  the  safraniu  a  cherry-red,  the 
inner  thickened  cells  a  rose-red.  The  thickened  rind  cells 
suddenly  cease  and  there  follow  two  or  three  layers  of 
tangentially- elongated-polygonal  cells  colored  a  cherry- 
red.  These  cells  form  the  endoderm,  but  are  distributed 
in  several  laj'ers,  have  no  undulating  bands  and  are  not 
otherwise  characteristically  thiclvened.  But  on  the  other 
hand  they  are,  like  the  Cells  of  the  endoderm,  cuticularized 
and  withstand  the  action  of  sulphuric  acid  very  well.  AVe 
will  designate  this  envelope  ot  cells,  vi,  the  inner  sheath. 
Next  follow  several  layers  of  likewise  wide  cells,  which 
often  contain  starch,  and  have  white  glittering  walls  ap- 
pearing as  if  swollen.  By  long  staining  they  are  colored 
an  orange-red.  These  cells  take  the  place  of  the  peri- 
cambium  and  may  therefore  as  in  the  ferns  be  called  peri- 
phloem,j92J.  Now  we  cometo  the  beautifully  stained  cherry- 
red  xylem  layer.  It  consists  of  wide  scaliform  vessels, 
sc,  separated  by  no  intervening  cells,  and  at  the  thin  edges 
of  protoxylem  elements,  that  is,  narrow,  ring  and  spiral 
vessels,  sp.  In  this  species  the  wood  layers  run  across 
the  cylinder  and  more  or  less  parallel  to  each  other. 
They  are  somewhat  concave  on  one  side  and  convex  on  the 
other,  and  one  can  see  by  reference  to  the  stem  in  its  pro- 
cumbent position  that  those  stripes  which  were  parallel  to 
the  surface  of  the  ground  had  their  concave  sides  turned 
upward.  The  small  vascular  liundles  ot  the  leaves,  when 
they  enter  the  central  cylinder,  are  joined,  as  in  the  ferns, 
to  the  group  of  spiral  vessels  of  a  wood  laj^er.  The  wood 
stripes  often  anastomose  as  in  the  lower  ones  of  the  illus- 
tration.    In  the  elongated  stem  of  the  Lycojjodium  selago 


144  VASCULAR   BUNDLES    OF   LYCOPODIUM. 

all  the  wood  stripes  are  iiiiitcd  and  form  a  star.  The.se 
wood  elements  are  surrounded  by  a  single  layer  of  thin- 
walled  narrow  cells  which  we  will  designate  the  wood  pa- 
renchyma, as  in  the  ferns.  On  the  ends,  they  extend  with 
their  protoxy lern  and  wood-parenchyma  to  the  protophloem 
tissue.  Between  the  wood  bands  is  the  bast,  the  larger 
cells  being  the  sieve-tubes,  v.  The  cells  are  white,  with 
strongly  refractive  walls,  narrow,  the  middle  row  only 
being  somewhat  Avider.  In  a  good  staining,  the  walls  of 
the  sieve-tubes  are  colored  a  rose-red,  while  the  rest  of 
the  bast  is  colorless.  On  the  edges  of  these  bands  of  sieve- 
tubes  are  the  nnrrow  protophloem  elements.  The  sieve- 
tubes  reach  the  periphloem,  by  these  protophloem  cells,  the 
essentially  wider  cells  of  the  former  being  clearly  distin- 
guished from  the  wood  and  bast.  In  cutting  the  section, 
the  inner  bast  and  wood  elements  of  the  vascular-bundle 
cylinder  are  easily  separated  from  the  rest  of  the  tissue  at 
the  inner  edge  of  the  protophloem. 

The  longitudinal  section  shows  us  first  the  epidermis 
and  next  the  rind  cells  running  diagonally  towards  it. 
Next  the  sclcrenehyma  fibres  of  the  outer  sheath  ;  and 
then  the  elongated  parenchyma  of  the  inner  sheath  ;  the 
periphloem  with  white  thick  walls  and  diagonally-placed 
partition  walls ;  the  scaliform  vessels  and  the  narrow, 
for  the  most  part  much-extended  ring  and  spiral  vessels, 
and  finally  the  bast.  The  latter  consists  of  very  long  cells 
which  join  each  other  at  the  ends  with  more  or  less  diag- 
onally-placed walls.  By  the  help  of  coralline  or  aniline 
blue,  we  may  recognize  the  small  inclined  sieve-plates. 
Only  the  wide  cells  in  the  bast  are  sieve- tubes  ;  the  more 
numerous,  narrow  cells  with  sparkling  granular  contents 
are  conducting-cells. 

Notes. 
(1)  See  De  Bary,  Comparative  Anatomy,  p.  170. 


LESSON    XIV. 
Cork.    Lenticels. 

We  have  already  had  an  opportunity  in  various  objects 
to  learn  something  of  the  nature  and  structure  of  cork. 
Nevertheless,  we  will  now  direct  our  particular  attention 
to  this  object  and  learn  to  know  the  lenticels  on  the  one 
side,  and,  on  the  other,  the  cork  cell  walls  and  their  reac- 
tions. 

A  cross-section  through  a  branch  of  Satnbiicus  nigra, 
about  3.^mm.  thick,  shows  us  the  vascular  bundles  distrib- 
uted in  the  medullary  crown,  about  the  wide,  large-celled 
pith,  already  united  by  interfascicular  cambium.  The 
cambium  has  already  begun  its  activity,  and  is  now  form- 
ing in  the  usual  way,  secondary  bast  without,  and  second- 
ary wood  within,  both  in  the  vascular  bundles  and  in  the 
interfascicular  spaces.  The  primary  bast  is  supported  on 
the  outside  by  sclerenchyma  fibres.  The  rind  is  from  ten 
to  fifteen  cells  thick.  The  projecting  edges  of  the  stem 
have  a  thick,  hypodermal,  collenchyma  layer,  which  in  the 
furrows  is  reduced  to  a  layer  three  or  four  cells  thick. 
The  collenchyma  layer  is  perforated  at  the  stomata  by  the 
green,  parenchyma  cells  of  the  rind  penetrating  through  to 
the  epidermis.  In  a  stem,  4  mm.  in  diameter,  the  cork  for- 
mation has  already  begun  by  the  tangential  division  of  the 
collenchyma  cells  immediately  bordering  on  the  epidermis. 
The  inner  of  the  two  sister  cells  again  divides,  and  the 
middle  cell  thus  formed,  subsequently  acts  as  the  cork  cam- 
bium cell.     This  is  easily  made  out  after  the  periderm  has 

10  {li5) 


146 


CORK    CELLS. 


become  several  cells  thick,  Fig.  58,  ph.  The  outermost, 
in  each  series  of  cells,  is  the  outer  part,  and  the  innermost 
the  inner  part,  of  the  original  collenchyma  cell,  d;  the  flat 
cell  next  to  the  inner  part,  ^/i,  is  the  cork-cambium  or  phel- 
logenic  cell.  In  a  favorable  section,  one  may  see  a  curious 
incident  in  the  formation  of  a  continuous  cork  layer  which 
begins  under  the  stomata.  The  primary  rind  cells  which 
surround  the  breathiug  cavity  begin  to  divide,  and  the 

division  reaches  over  into 
the  adjoining  collenchyma 
cells.  Soon,  under  the 
stoma,  a  meniscous-shaped 
layer  of  dividing  cells  is 
formed,  Fig.  59,  ^?,  which 
produce  towards  the  surface 
colorless  oblong  cells,  Z, 
and  towards  the  inside  cork- 
rind  cells,  jpd  (phello- 
derma).  The  upper  are 
designated  "filling"  cells. 
They  take  a  brown  color, 
but  are  not  suberized,  and 
FIG.  58.  Transection  through  the  upper   ^y  their  numerical  increase 

smi'ace  of  a  young  stem  of  Samhucus  ni-  ,  .  ,  . 

^m  epidermis,    ph,  phellogeu;  cl  and  ci,  SO  ^:reSS  Upon  the  CpidcrmiS 

the  upper  and  under  parts  of  the  original  ^g  ^^  mpture  it.  ThuS  are 
collenchyma  cells.  X  240,  ^ 

the  rind  pores  or  lenticels 
produced.  Examined  with  the  naked  eye,  the  lenticels 
appear  to  be  furrows  surrounded  by  two  lip-like  pads. 
The  brown  color  of  the  filling  cells  is  quite  apparent.  On 
the  younger  places  of  the  stem,  the  lenticels  appear  as 
oblong  swollen  spots.  Still  younger  stages  are  indicated 
by  a  somewhat  brighter  color.  The  section  should  be 
made  through  these  places  in  order  to  show  the  earliest 
stages  of  development.     Directly  after  the  rupturing  of 


LENTICELS. 


147 


the  epidermis,  the  division  of  the  colleiichyma  cells  begins, 
which  leads  to  the  formation  of  the  periderm.  The  filling 
cells  of  the  lenticels  separate  from  each  other ;  but,  as  they 
disorganize  from  without,  the  cambium  builds  them  up 
from  beneath.  Tiie  spaces  between  the  filling  cells  are 
filled  with  air,  and  thus  the  atmospheric  air  is  admitted  to 
the  inner  tissue  of  the  stem.  It  thus  takes  the  place  of 
the  stomata  in  those  older  parts  of  the  stem  in  which  cork 
has  begun.  For  the  winter,  somewhat  closer  formation 
and  more  resistent  fillinsf  cells  are  formed. 


pd  y 


Fig.  59.    Transection  of  a  lenticel  of  Savibucus  nigra,    e,  epidermis;  ^j/t,  phel- 
logen;  I,  filling  cells ;;}?,  cambium  of  lenticel ;  ;;rf,  phelloderm.  X  90. 

An  essential  enveloping  layer  for  the  winter  time  formed 
of  narrow  cells,  which  touch  each  other,  is  not  found  in 
Sambucus,  as  in  many  other  plants.  But  the  intermediate 
laj'^ers  which  are  intercalated  between  the  filling  cells  from 
time  to  time  serve  the  same  purpose.  The  cells  of  these 
closing  and  interstitial  layers  are  suberized,  but  have  in- 
tercellular spaces  between  them,  so  that  the  closure  is  not 
quite  perfect  (2).  In  old  stems  of  Sambucus,  the  peri- 
derm is  ruptured  longitudinally.     These  clefts  go  through 


148  CORK   CELLS. 

the  lenticels  without  injuring  them.  The  latter  are  pre- 
served still  in  quite  old  stenas,  while  the  outer  periderm 
layers  are  interleaved  between  them. 

We  Avill  next  take  the  Cyiisus  laburnum  for  the  study 
of  the  structure  of  cork  cells  as  they  are  remarkably 
thickened  in  this  species.  A  cross-section  through  the 
rind  of  an  old  stem  shows  the  periderm  constructed  of 
but  one  kind  of  cork  cells  regularly  arranged  in  a  radial 
series.  The  youngest  cork  cells  are  colorless,  the  older 
yellow,  and  the  oldest  a  yellow-brown.  The  outside  cells 
are  tangential  ly  elongated  even  to  the  closing  up  of  the  cell 
cavity.  All  these  cork  cells  are  much  thickened,  espec- 
ially on  the  outside.  Without  the  help  of  reagents,  the 
delicate  middle  lamella  is  easily  distinguished,  also  a  stout 
clearly  unlaminated,  secondary,  thickening  layer,  and  on 
the  inside  of  the  latter  a  tertiary  thickening  layer.  So 
each  wall,  which  separates  any  two  cells,  consists  of  at 
least  five  layers  :  the  middle  lamella  which  here  represents 
the  primary  wall  and  is  lignified  ;  the  two  secondary  thick- 
ening layers  which  are  alone  suberized ;  and  the  two  ter- 
tiary thickening  layers,  which  often  betray  their  cellulose 
character  and  may  be  designated  the  cellulose  layer,  but 
in  this  case  are  a  little  lignified.  Chloriodide  of  zinc  colors 
the  cork  cells  yellow  to  yellow-brown,  the  younger  darker 
than  the  older,  the  tertiary  layers  the  darkest.  Char- 
acteristic reactions  on  cork  substance  or  suberine  are 
those  of  potash,  the  maceration  mixture  and  chromic  acid 
(3).  Treat  the  section  with  potash  lye  and  notice  that 
the  cork  cells  become  yellow.  Careful  warming  on  the 
slide  under  the  cover-glass  increases  the  intensity  of  the 
color.  With  the  maceration  mixture  (chloric  acid,  potash 
and  nitric  acid)  we  get  the  eerie  acid  reaction.  The  cold 
mixture  brings  out  all  the  parts  of  the  cork  cell  in  a  yellow- 
brown  color.     Heat  the  preparation  with  an  added  quantity 


CORK    CELL    REACTIONS.  149 

of  the  reagent,  when  necessary,  and  the  whole  section  will 
be  dissolved  except  the  suberized  membrane.  The  color- 
less globular  masses  which  remain  are  the  so-called  eerie 
acid,  soluble  in  alcohol,  but  much  more  easily  in  ether. 
Strong  chromic  acid  dissolves  the  whole  section  except 
the  suberized  layer  of  the  cork  cells,  and  after  a  while 
this  becomes  so  transparent  as  to  be  difficult  to  find.  Still 
it  does  not  disappear.  Although  the  middle  lamella  has 
been  dissolved,  the  secondary  thickening  layers  still  hang 
together. 

The  common  flask  cork  (from  Quercus  suber)  consists  of 
almost  cubical,  thin- walled,  relatively  large  cells,  which  are 
commonly  somewhat  thicker  and  flatter  near  the  limit  of 
the  year's  production,  and  are  succeeded  by  the  cubical 
cells  again.  Potash  colors  the  section  yellow  especially 
the  thick-walled  cells.  The  five  layers  of  the  double  walls 
between  the  cells  are  traceable  as  in  the  last  specimen. 
The  tertiary  layer  does  not  give  the  cellulose  reaction  at 
first,  but  only  after  proper  treatment.  The  suberine  reac- 
tions are  even  more  beautiful  than  in  Cytlsus  especially  the 
eerie  acid  reaction. 

It  often  happens  that  from  the  phellogen  not  alone 
centrifugal  cork  cells,  but  also  centripetal  rind  cells,  are 
formed,  the  so-called  "phelloderm." 

The  phelloderm  seldom  reaches  the  thickness  that  it 
has  in  the  Rihes  species.  Prepare  a  cross-section  through 
an  old  stem  of  lithes  rubrum,  and  we  shall  find  under 
the  thin-walled,  brown  cork-layer,  first,  the  phellogen 
and  then  a  thick  layer  of  flat  riud  cells  containing  chloro- 
phyll ;  the  latter  ari3  arranged  in  radial  rows  which  co- 
incide with  those  of  the  adjoining  cork.  In  the  inner 
parts  of  the  phelloderm,  the  radial  arrangement  is  lost  in 
consequence  of  the  supplementary  extension.  The  inner- 
most phelloderm  cells  adjoin  the  collenchyma  of  the  riud. 


150  CORK    CELLS. 

All  those  formations  which  arise  from  the  phellogen  are 
included  in  the  term  periderm.  In  Hibes,  therefore,  the 
periderm  is  formed  of  cork  (phellem)  and  cork  rind 
(phelloderm) .  It  is  interesting  to  make  a  section  through 
a  this  year's  stem  of  Hibes  rubrum^  in  which  the  cork 
formation  has  but  just  begun.  We  shall  see  the  first  be- 
ginnings of  the  formation  of  the  phelloderm,  and  perhaps, 
demonstrate  that  in  this  plant  the  phellogen  is  pretty 
deeply  embedded  in  the  rind.  The  extreme  outside  por- 
tions, being  cut  off  from  the  sap-bearing  tissue  by  the  cork 
layer,  soon  die,  become  brown  and  are  thrown  ofi"  as  the 
so-called  bark. 

Notes. 

(1)  Literature  in  de  Bary,  Vergl.  Auat.  p.  560;  v.  Höhnel,  Stzber. 
d.  math,  naturw.  CI.  d.  k.  Ak.  d.  W.  in  Wien,  Bd.  lxxvi,  1877. 

(2)  Klebahu,  Jen.  Zeitschr.  f.  Naturw.,  Bd.  xvii. 

(3)  Introduced  by  v.  Höhnel.  Work  and  Vol.  quoted  above,  p.  522. 


LESSON  XV. 
Structure  of  the  Foliage  and  Floral  Leaves.     The 

ENDS   OF   THE    VaSCULAR   BuNDLES. 

We  shall  now  take  a  series  of  objects  which  will  make 
us  acquainted  with  the  structure  of  leaves.  We  shall  be- 
gin with  the  foliage  leaves  and  take  those  forms  which  will 
show  us  the  widest  possible  diiferences  in  the  inner  struct- 
ure of  the  leaves.     The  first  example  shall  be  Ruta  grav- 


FiG.  60.  Leaf  epidermis  and  adjoining  tissue  of  fiwio  gTrtreotens.  ^,  epidermis 
of  the  upper  side;  sc,  epidermis  cells  over  a  secretory  receptacle;  pa,  palisade  pa- 
renchyma; B,  epidermis  of  the  under  side;  s,  spoage-parenchyma.  In  A,  the  arc 
filled  spaces  are  shaded;  in  B  they  are  clear. 

eolens  whose  leaves  are  mostly  retained  through  the 
winter.  The  leaves  are  doubly  pinnated,  the  leaflets  a  re- 
verted ovate.  Held  towards  the  light  clear  jDoints  are  seen 
in  the  leaf.  They  are  reservoirs  of  essential  oil ;  "  inner 
glands"  in  the  tissue  of  the  leaf.  We  make  first  a  super- 
ficial section  of  the  epidermis  and  observe  first  that  the 
upper  side,  Fig.  60,  A,  has  but  few  if  any  stomata,  while 

(151) 


152  STRUCTURE    OF   FOLIAGE    LEAF. 

there  are  many  on  the  under  side,  Fig.  60,  B.  Both 
on  the  upper  and  under  side  four  epidermal  cells  lie 
over  the  inner  gland,  A,  sc,  somewhat  depressed  m  the 
middle.  In  thicker  parts  of  the  section,  where  the  reser- 
voir has  not  been  cut  by  the  knife,  one  may  find  a  yellow 
strongly  refractive  drop  of  matter.  By  deeper  focussing 
one  may  see  that  the  tissue  of  the  upper  side  of  the  leaf 
consists  of  cells  whose  optical  section  is  round,  A, p. 
These  cells  are  laterally  almost  entirely  separated  from 
each  other  and  the  intercellular  spaces  filled  with  air. 
On  the  epidermis  of  the  under  side,  cells  with  alike  round 
optical  section  are  seen  but  in  much  smaller  number,  B,  s. 
These  cells  also  are  separated  with  air,  and  free  wide  breath- 
ing cavities  are  seen  under  the  stomata  B.  Now  make  a 
transverse  section  perpendicular  to  the  longer  axis  of  the 
leaflet,  using  a  piece  of  elder-pith  as  already  described  for 
makinof  the  section.  This  section  will  show  us  the  leaf- 
tissue  or  the  "mesophyll."  First,  beneath  the  upper  epi- 
dermis. Fig.  61,  ep',  are  the  "palisade  cells," ^?',  a  double 
layer  of  parallel  elongated  cells  containing  chlorophyll, 
perpendicular  to  the  surface  of  the  lejif.  We  have  seen 
that  these  were  laterally  somewhat  separated  from  each 
other,  but  we  find  that  the  cells  of  the  two  layers  are 
joined  fast  together  at  their  ends.  The  cells  of  the  sec- 
ond palisade  layer,  pi/'  are  somewhat  less  numerous 
than  those  of  the  first,  and  (jften  two  of  the  outer  are  united 
to  one  of  the  latter.  Next  to  these  layers  succeeds  a 
loose  tissue  of  cells  which  forms  a  network  with  open 
meshes  which  extends  quite  to  the  under  epidermis.  We 
call  this  tissue  the  "sponge-parenchyma."  It  has  some- 
what fewer  chlorophyll  grains  than  the  palisade  tissue. 
The  cells  of  the  upper  layer  of  sponge-parenchyma,  sp', 
are  connected  with  the  inner  layer  of  palisade  cells  and 
indeed  are  united  to  a  larger  luunber  of  palisade  cells. 


LEAF    STRUCTURE. 


153 


Fig.  61.  Transection  throiigli  the  leaf  of  Riäa  graveolens.  ep',  epidermis  of  the 
tipper  side;  e/?",  of  the  under  side;  pr,  pr', palisade  parenchyma;  sj),  sponge-paren- 
chyma; /.-,  crystal-bearing  cell;  j'.s,  vascular  bundle;  sc,  secretion  receptacle;  a, 
breathing  cavity;  si,  stoma  ta.  X  210. 


154  LEAF   STRUCTURE. 

None  of  the  palisade  cells  are  free  on  their  lower  ends. 
When  they  seem  to  be,  as  in  some  cases  in  the  illustration, 
it  is  only  that  their  connecting  cells  are  not  in  the  plane  of 
the  image.  So  also  in  the  sponge  tissue  there  are  no  cells 
with  free  ends,  but  all  are  united  at  their  ends.  The  un- 
der layer  of  sponge-parenchyma,  sp'",  extends  to  the  epi- 
dermis and  joins  it  more  or  less  perpendicularly,  thus 
giving  us  a  form  of  tissue  intermediate  between  the  pali- 
sade and  the  sponge  tissue.  The  breathing  spaces,  a,  nnder 
the  stomata,  st^  &:e  left  free.  Crystal  masses  of  calcium 
oxalate,  k,  are  found  in  some  of  the  cells.  These  cells 
are  swollen,  tun-shaped,  contain  no  chlorophyll  and  seem 
to  be  suspended  between  the  green  cells.  At  the  edges  of 
the  leaflets  the  epidermis  cells  are  greatly  thickened  on  the 
outside,  the  palisade  layers  are  reduced  to  one  and  grad- 
ually change  over  into  the  elongated  sponge-parenchyma 
layer  of  the  under  side  of  the  leaf,  sp'".  The  vascular 
bundles  lie  in  the  sponge-parenchyma ;  the  largest,  the 
middle  nerve  of  the  leaflet,  extends  on  the  one  side  almost 
to  the  inner  palisade  layer  and  on  the  other  to  the  lowest 
extended  sponge-parenchyma  layer.  In  the  vascular  bun- 
dle itself  we  may  easily  recognize  in  the  darker  part  the 
vessels  and  in  the  brighter  the  bast.  The  radial  arrange- 
ment of  these  elements  assures  lis  of  the  activity  of  the 
cambium  at  some  time.  About  the  vascular  bundle  is  a 
parenchyma  sheath  whose  cells  contain  chlorophyll  grains 
and  to  the  outer  of  which  the  sponge-parenchyma  cells 
are  attached.  The  same  relations  hold  in  the  smaller  vas- 
cular bundles  represented  in  the  illustration.  Still  smaller 
bundles,  vs,  which  have  but  few  vessels  and  bast  cells,  are 
met  with  in  the  transverse  section.  They  are  to  the  last 
still  surrounded  with  a  sheath  of  elongated  parenchyma 
cells.  The  secretion  reservoirs,  sc,  touch  the  upper  or  un- 
der epidermis.     They  are  circular  in  outline,  inclosed  by 


LEAF    STRUCTURE.  155 

a  layer  of  thin-Avalled  more  or  less  disorganized  cells, 
upon  which  borders  a  layer  ©f  flat  cells  havmg  tolerably 
strong,  white  walls  and  granular  contents.  Adjacent  to 
these  cells  is  the  mesophyll  with  its  chlorophyll  contents. 
The  epidermal  cells  which  overlie  the  reservoir  are  flat 
like  those  which  surround  it.  The  volatile  oil  may  be  re- 
moved by  alcohol.  A  superficial  section  made  at  the  base 
of  the  common  petiole  shows  that  the  epidermal  cells  are 
elongated  and  on  the  upper  as  well  as  the  under  side  are 
interrupted  Avith  stomata.  The  oil  reservoir  is  also  not 
wanting.  Under  the  epidermis  is  a  layer  of  elongated 
collenchvma  cells  and  next  to  that  the  tissue  containino: 
chlorophyll.  The  transection  of  the  petiole  shows  the 
epidermis  thickened  on  the  outside  ;  beneath  this  the  sim- 
ple layer  of  thickened  collench^-ma  cells,  which  is  inter- 
rupted only  by  the  stomata.  The  two  or  three  layers  of 
elongated,  green,  palisade  cells  are  quite  uniformly  de- 
veloped in  the  whole  circumference,  but  are  rather  looser 
on  the  under  side.  Within  these  are,  finally,  round,  first 
green,  then  colorless  cells  which  become  larger  toward 
the  middle.  In  this  inner  cylinder,  in  colorless  cells,  run 
the  vascular  bundles,  the  largest  in  the  middle  nearer  the 
under  side,  the  others  in  the  circumference,  diminished 
in  size  on  both  sides  and  with  their  wood  parts  turned 
towards  the  middle  of  the  petiole.  The  larger  of  these 
bundles  are  provided  with  strings  of  sclerenchyma  fibres. 
Apparently  the  activity  of  the  cambium  has  been  more 
prolonged  in  these  vascular  bundles  which  has  produced 
secondary  wood  within  and  secondary  thin-walled  bast  with- 
out. The  larger  vessels  appear  only  in  the  inner  part  of 
the  vascular  bundles  and  the  border-pitted  tracheids  in  the 
outer  portions. 

We  will  now  take  a  leaf  of  jTcrgus  silvaiica  for  our  in- 
vestigation.    On  account  of  the  greater  thinness  of  the 


156 


LEAF   STRUCTURE. 


leaf  it  is  less  easy  to  get  a  sufficiently  thin  section.  Take 
therefore  a  very  small  piece  of  the  leaf  between  the  two 
pieces  of  elder-pith.  Stomata  are  found  only  on  the  un- 
der side.  Attached  to  the  epidermis,  ejo,  Fig.  62,  of  the 
upper  side,  in  leaves  from  f- mny  localities,  is  a  layer  of 
elongated,  palisade  cells,  pi,  which  are  more  or  less  sep- 
arated from  each  other  by  intercellular  spaces.  They  are 
grouped  together  in  bundles  below,  and  each  bundle  sets 
on  one  or  more  funnel-shaped,  broadened,  sponge-paren- 
chyma cells,  sp'.    The  latter  are  c(mnected  into  a  network 


Fig.  (i2.  Transection  of  a  leaf  of  Fagus  silvatica.  ep,  epidermis ;  pi,  palisade 
parenchyma;  sp.  sponge-parenchyma;  k,  crystal  bearing  cells;  iu  k',  a  cluster  of 
crystals;  st,  stoma.  X  ^60. 


by  elongated,  sponge-parenchyma  cells,  which  reaches  to 
the  epidermis  of  the  underside,  ep'\  Single  cells  without 
chlorophyll,  but  with  crystal  clusters  in  them,  k',  are  em- 
bedded in  the  sponge-parenchyma.  The  principal  nerve 
and  the  lateral  nerves  of  the  first  order  project  from  the 
under  side  of  the  leaf  as  leaf  ribs.  The  projecting  por- 
tion of  the  nerve  is  about  as  thick  as  the  rest  part  of  the 
leaf.  The  vascular  bundles  extend  into  the  projecting  ril). 
The   latter   is   covered    with    elongated,   epidermal   cells 


LEAF   STRUCTURE.  157 

which  are  followed  by  elongated,  collenchyma  cells.  To 
these  succeed  cells,  each  of  which  bears  a  simple  crystal, 
and  then  the  many-layered  sheath  of  sclerenchyma  fibres 
\/hich  encloses  the  whole  vascular  bundle.  On  the  upper 
side,  over  the  vascular  bundle,  the  palisade  layer  is  in- 
terrupted in  a  narrow  place  and  is  replaced  by  collenchyma 
on  which  follows  a  slender  stripe  of  elongated,  epidermal 
cells,  ep'".  A  layer  of  cells,  containing  chlorophyll,  en- 
closes the  sclerenchyma  sheath  and  on  them  are  the  sponge- 
parenchyma. 

The  ribs  represent  the  mechanical  system  of  the  leaf 
which  must  be  built  in  conformity  to  that.  The  filaments 
are  uniformly  apparent  in  the  surface  of  the  leaf,  the 
plane  of  the  filaments  being  perpendicular  to  that  of  the  leaf. 
The  upper  surface  of  the  leaf  is  principally  stretched  by 
pulling,  and  the  under  surface  by  pressure.  The  fila- 
ments in  th  present  case  are  I-shaped,  the  vascular  bun- 
dles forming  the  filling  of  the  filaments.  The  stiffness  of 
the  under  girding  depends  greatly  upon  their  springing 
as  far  as  possible  below  the  under  surface  of  the  leaf  from 
the  projecting  midrib.  The  nerves  expand  the  blade  of 
the  leaf,  give  it  the  necessary  stiffness  and  stability  and 
prevent  its  being  torn. 

Small  vascular  bundles,  like  those  in  the  illustration, 
are  supported  only  on  the  upper  and  under  sides  with 
sclerenchyma  fibres.  The  branches  of  these  are  without  a 
sclerenchyma  layer  embedded  directly  in  the  parenchyma. 
The  smaller  vascular  bundles  are  accompanied  both  on  the 
bast  and  wood  parts  by  crystal-bearing  cells,  k.  Above 
and  beneath  it  the  epidermal  cells  are  somewhat  extended 
and  form  shallow,  depressed  stripes.  Long  hairs  of  scler- 
enchyma fibres  grow  from  the  epidermal  cells  over  the 
nerves,  but  fall  away  from  the  full-grown  leaves. 

Leaves,  growing  in  sunny  situations,  are  thicker  than 


158  LEAF   STRUCTURE. 

those  growing  in  deep  shadows  (2) .  The  additional  thick- 
ening comes  from  an  elongation  of  the  palisade  paren- 
chyma cells  and  an  increase  of  the  number  of  the  layers. 
The  palisade  tissue  is  thus  adapted  to  greater  intensity  of 
light  and  the  sponge-parenchyma  to  a  less.  In  palisade 
cells  the  chlorophyll  grains  are  seen  only  in  profile,  on  the 
elongated  side  walls,  protruding  less  or  more  according  to 
the  intensity  of  the  illumination  into  the  cell  cavity.  On 
the  contrary,  the  chlorophyll  grains  of  the  sponge-paren- 
chyma are  seen  in  profile  or  on  the  surface  according  to 
the  intensity  of  the  illumination  ;  that  is,  they  take  a  posi- 
tion parallel  or  perpendicular  to  the  upper  surface  of  the 
leaf.  The  palisade  cells  first  receive  the  rays  of  light, 
while  the  sponge-parenchyma  cells  receive  it  only  after 
it  has  been  weakened  by  absorption  in  passing  through 
the  palisade  cells.  This  disadvantage  is  in  part  compen- 
sated for  by  the  sponge-parenchyma  cells,  exposing  the 
greatest  possible  amount  of  surface  to  it.  But  if  the  light 
become  too  intense,  the  chlorophyll  grains  turn  their  edges 
to  it.  Such  leaves  which  are  developed  in  the  bright 
sunlight  are  composed  almost  entirely  of  palisade  cells, 
while  those,  only  about  one-third  as  thick,  grown  in 
deep  shadow,  consist  almost  exclusively  of  sponge-paren- 
chyma. 

Still  other  physiological  consideration  will  be  connected 
with  our  morphological  investigations  whose  correctness 
may  be  tested  by  the  microscopic  image. 

The  assimilation  of  carbon  takes  place  in  definitely  col- 
ored chromatophores  and  in  the  higher  plants,  exclusively 
in  the  chlorophyll  grains.  Only  these  plasma  bodies  have 
the  capacity,  in  light  of  sufficient  intensity,  to  disintegrate 
the  atoms  of  water  and  carbonic  dioxide  and  form  from 
them  combinations  which  are  rich  in  carbon.  This  proc- 
ess, taking  place  mainly  in  the  palisade  cells,  requires  us 


LEAF   STRUCTURE.  159 

to  designate  them  physiologically  as  the  principal  assimi- 
lation cells.  The  palisade  cells,  as  we  have  already  seen, 
are  more  or  less  fully,  laterally,  separated  from  each 
other,  and  below  bend  together  into  tufts.  So  the  as- 
similated matter  will  not  pass  laterally  from  cell  to  cell, 
but  rather  into  the  widened  funnel-shaped  cells  of  the 
sponge-parenchyma  upon  which  the  tufts  of  palisade  cells 
rest,  sp'.  Figs.  61,  62,  the  physiological  function  of  which 
therefore  is  that  of  absorbeut  or  collecting:  cells.  From 
the  same  point  of  view  the  next  following  cells  of  the 
sponge-parenchyma,  sjt" ,  Figs.  60,  61,  may  be  designated 
conducting  cells.  Since  the  sponge-parenchyma  has  wide 
air  spaces  in  connection  with  the  stomata  it  may  be  desig- 
nated ventilation  tissue  ;  also  as  transpiration  tissue,  since 
a  considerable  evaporation  takes  place  from  the  surface  of 
the  cells  into  the  intercellular  spaces.  It  is,  on  account  or" 
its  chlorophyll  contents,  rightly  known  also  as  assimilation 
tissue.  The  sponge-parenchyma  cells  are  directly  attached 
to  the  parenchyma  sheaths  of  the  vascular  bundles  and  so 
c  >nduct  the  assimilated  material  partly  to  that  and  partly 
to  the  bast  of  the  vascular  bundle.  Sheath  and  bundle  to- 
gether, therefore,  are  conductors.  The  vascular  bundles 
also  conduct  water  from  the  woody  part  of  the  plant,  giv- 
ing it  out  into  the  surrounding  tissue  of  the  leaf,  part  of 
which  finds  its  way  into  that  water  reservoir,  the  epider- 
mis. The  conducting  tissue  of  the  parenchyma  sheath 
about  the  vascular  bundles,  nuich  thickened  and  giviuof 
solidity  to  the  "mechanical  cells,"  likewise  forms  the  tis- 
sue of  the  projecting  leaf  ribs  and  is  known  as  "nerve  par- 
enchyma." This  "nerve  parenchyma"  is  continued  into 
the  fundamental  tissue  of  the  petiole,  which,  as  we  have 
seen  in  the  Huia,  is  built  principally  of  conducting  and 
mechanical  elements.  Assimilating  cells  play  but  a  sub- 
ordinate part  in  it. 


160  LEAF   STRUCTURE. 

Let  US  now  study  the  iuner  structure  of  a  floral  leaf 
and  use  the  opportunity  also  to  learn  of  the  course  and 
ending  of  the  vascular  bundles.  Petals  of  Verbascmn  ni- 
grum are  especially  well  adapted  to  both  of  these  purposes. 
The  air  bubbles  which  adhere  to  the  petal  may  be  driven 
out  by  tapping  lightly  on  the  cover-glass.  Use  no  alco- 
hol. We  observ^e  a  delicate  epidermis  and  from  two  to 
four  layers  of  sponge-parenchyma,  two  at  the  edges  and 
four  in  the  thicker  part  of  the  petal.  The  stoutest  vascu- 
lar bundles,  as  Avell  as  the  finest  branches  where  they  are 
reduced  to  spiral  vessels,  are  sheathed  in  a  layer  of  elon- 
gated, thin-walled,  parenchyma  cells.  This  parenchyma 
sheath  closes  toofether  in  front  over  the  ends  of  the  vascu- 
lar  bundles.  Protoplasmic  streaming  may  be  seen  in  the 
cells.  The  stout-branched,  sponge-parenchyma  cells  are 
joined  to  the  elements  of  the  sheath.  Particularly  beau- 
tiful is  the  view  of  the  ends  of  the  bundles  which  exhibit 
a  radiating  juncture  of  the  sponge-parenchyma  cells  on 
the  sheath. 

The  petals  of  Pajpaver  Mhoeas  has  but  one  layer  of 
sponge-parenchyma  between  the  upper  and  under  epider- 
mis. The  vascular  bundles  never  end  free,  but  rather 
lock  together  in  commin2;led  arches  at  the  edo-es  of  the 
leaf.  They  are  surrounded  in  their  whole  course  by  a 
parenchyma  sheath  of  a  single  layer  of  cells,  to  which  the 
sponge  parenchyma  cells  are  joined  on  both  sides. 

Notes. 

(1)  See  Haberlandt,  iu  encykl.  d.  Naturwiss.,  Handb.  d.  Bot.,  Bd.  ii, 
p.  614;  J.  V.  Sachs,  Vorlesungen  über  Pflanzen-Physiologie,  p,  59  ff. 

(2)  See  Stahl,  zuletzt  Jeu.  Zeitschr.  f.  Naturvv.  Bd.  xvi,  1883; 
Concerning  the  influence  of  sunny  and  shady  locations  on  the  forma- 
tion of  the  foliage  leaves. 

(3)  See  Haberlandt,  work  and  vol.  quoted  above,  p.  G40. 


LESSON  XVI. 

The  Vegetative  Cone  of  the  Stem. 

Our  next  task  shall  be  to  select  some  suitable  object 
which  shall  make  us  acquainted  with  the  structure  of 
the  vegetative  point  of  the  vascular  plants.  We  choose 
the  phanerogam  Hippuris  vulgaris  (Ij  whose  vegetative 
cone  is  strongly  developed  and  easily  prepared.  Take  a 
thrifty  sprout  and  cut  oif  a  piece  from  the  top  about  a  cen- 
timeter long.  Remove  the  larsfer  leaves.  Now  take  the 
bud  between  the  thumb  and  forefinger,  holding  it  with  the 
top  down  and  with  a  razor  held  perpendicularly,  and  with  a 
drawing  motion,  cut  the  bud  longitudinally  exactly  in 
halves.  Now  take  one  of  the  halves  and  in  the  same  way 
halve  it,  then  the  half  of  this  lying  nearest  the  centre 
of  the  bud,  and  so  on  till  a  section  of  sufficient  tenuity  be 
obtained.  This  jnanipulation  may  not  at  first  be  success- 
ful, but  it  is  not  a  matter  of  any  great  difficulty  and  a  little 
practice  ought  to  make  it  easy  enough. 

If,  however,  one  does  not  overcome  the  difficulty,  he 
may  hold  the  severed  bud  between  two  flat  pieces  of  elder 
pith  and  cut  as  he  did  between  the  thumb  and  finger,  but 
hitting  the  right  point  in  the  object  will  be  much  more  a 
matter  of  chance  in  this  case.  But  objects  of  this  kind 
may  also  be  fastened  between  the  edges  of  two  pieces  of 
elder  pith  and  the  cut  made  through  them  and  the  pith  at 
the  same  time,  as  already  explained. 

Select  a  section  from  the  exact  middle   of  the    bud, 

which  we  recognize  by  the  slender  regularly-constructed 

vegetative  cone.     This  vegetative  cone  forms  the  leaves 

in  a  many-branched  whorl,  which  may  be  seen  at  some  dis- 

11  (IGl) 


162  VEGETATIVE    CONE    OF    STEM. 

tuiice  from  the  top  as  isolated  knobs  set  uniformly  about 
its  circumference.  Beneath  the  youngest  Avhorl  but  one,  the 
node  of  the  stem  begins  to  be  indicated  by  a  transverse, 
thick  tissue  plate,  above  and  below  which,  in  the  rind  of 
the  stem,  the  air  passages  enter.  These  air  passages, 
which  extend  from  one  node  to  another,  increase  in  size 
with  the  growth  of  the  stem.  The  internodes  grow  rap- 
idly and  uniformly,  both  in  length  and  thickness.  The 
vessels  of  the  stem  beoin  to  form  somewhat  below  the 
fourth  youngest  whorl  of  leaves.  The  addition  of  a  little 
potash  lye  brings  them  out  very  finely.  These  vessels 
appear  in  the  longitudinal  axis  of  the  stem  to  belong  to  the 
vascular  bundle  which  grows  at  the  extremity  and  ends  at 
the  top  in  a  single  ring  vessel.  The  vessels  which  belong 
to  the  leaves  make  their  appearance  iirst  in  the  tenth  or 
twelfth  whorl,  and  are  joined  to  the  vessels  of  the  vascu- 
lar bundle  of  the  stem.  At  a  point  not  so  far  removed 
from  the  apex,  little  flat  knobs  begin  to  appear  in  the  axils 
of  the  leaves  which  are  the  beginnings  of  fan-shaped  scales 
borne  on  simple,  short  stile-cells.  Only  in  plants  taken 
in  their  blooming  season  do  we  find  here  the  axillary  buds. 
In  order  to  study  thoroughly  the  structure  of  the  vegeta- 
tive cone,  we  should  select  a  good  median  longitudinal 
section,  treat  it  with  concentrated  potash  lye  and  having 
washed  it,  lay  it  in  concentrated  acetic  acid.  After  a  little 
while  examine  it  in  the  same  or  in  potassium  acetate.  It 
may  be  handled  to  best  advantage,  since  we  wish  to  ex- 
amine both  sides  of  it,  by  putting  it  between  two  cover 
glasses  and  then  laying  these  on  a  slide,  but  with  no  fluid 
between  the  lower  one  and  the  slide.  It  can  then  be 
turned  over  very  readily.  By  strong  magnification  we  ob- 
serve a  definite  arrano-ement  of  the  cells  in  the  "meristem" 
of  the  vegetative  cone.  See  Fi«:.  63.  There  are  mantel- 
like  layers  of  cells  whose  division  walls  form  a  band  of 


VEGETATIVE  CONE  OF  STEM. 


163 


confocal  parabolas.  The  outer  layer  which  runs  over  the 
foundations  of  the  leaves  and  covers  the.  whole  cone  is 
the  derraatogen,  d,  and  forms  the  epidermis.  Under  this 
there  are  four  or  more  undiflerentiated  layers  of  tissue 
which  belong  to  the  periblem,^?-,  out  of  which  the  rind  of 
the  stem  is  developed.  Finally,  we  come  to  a  central 
cylinder  with  a  reduced  cone  at  top,  which  mostly  ends  in 
a  single  cell,  and  out  of  which,  as  we  shall  see,  by  looking 
deeper  into  the  section,  is  formed  the  vascular  bundle  in 
the  axis  of  the  stem.  This 
tissue  we  call  the  plerome, 
^l.  Thus  the  epidermis, 
rind  and  vascular  bundle  of 
the  stem  in  Hij^puris  have 
their  own  "  h  i  s  t  o  g e  n." 
There  is,  indeed,  no  single 
apical  cell  but  each  histo- 
gen  ends  at  the  top  of  the 
veofetative  cone  in  one  or 
more  "initial"  cells. 

It  must  be  added  that  in 
all  phaneroo-ams,  the  sep-     „_  „„    ^      ■,  -,■^     ,■      p^, 

t^  o  '  i  Fig.  63.    Longitudinal  section  of  the  veg- 

aration  of  the  histOgenS  in  etatlve  cone  of  mppurls  vulgaris,  d,  der- 
.,  ...  .    1  inatogen;  pr,  periblem ;  pi,   plerome:  /, 

the  vegetative  cone  is  by  no  beginning  of  the  leai.  x  240. 
means  so  distinctly  marked 

as  in  this  case.  In  many  gymnosperms,  Abieiinece,  Gy- 
cadea,  there  is  no  sharp  demarcation  between  the  dermat- 
ogen  and  the  periblem  and  often  also  the  periblem  and 
plerome  are  not  distinctly  separated.  In  the  angiosperms 
the  dermatogen  is  always  distinctly  set  oif,  but  there  often 
exists  no  boundary  between  the  periblem  and  plerome. 
It  is  not  in  general  a  difference  of  tissue  which  is  continued 
into  the  meristem  of  the  cone  that  gives  the  necessary  sta- 
bility to  the  young  tissue,  but  rather  the  mechanical  ar- 


164  VEGETATIVE    CONE   OF   STEM. 

rangement  of  the  cell  walls.  We  meet,  in  this  arrange- 
ment of  the  cells,  the  two  sorts  of  cell  division :  the  anti- 
clinal, that  is,  perpendicular  to  the  outer  surface  of  the 
plant,  and  the  periclinal,  a  division  of  cells  parallel  to  that 
surface  (2). 

We  may  retain  the  terms  dermatogen,  periblem  and 
plerome,  in  all  cases,  because  the  arrangement  of  cell-layers 
which  we  have  observed  in  Uippuris  frequently  recurs  in 
phanerogams,  and  these  terms  will  serve  to  designate, 
therefore,  definite  regions  of  the  vegetative  cone.  The 
epidermis  really  arises,  in  the  angiosperms,  only  from  the 
dermatogen.  But  the  vascular  bundles  may  not  always 
find  their  origin  in  the  plerome,  but  also  in  the  periblem. 
In  the  origination  of  the  leaves  we  see,  as  in  Fig.  63,  first 
a  periclinal  parting  of  the  cells  and  then  an  anticlinal,  in 
the  outer  layer  of  the  periblem.  The  dermatogen  remains 
a  single  layer  even  over  the  arched  places,  and  has  only 
an  anticlinal  cell-parting.  An  anticlinal  and  periclinal 
division  of  cells  takes  place  in  the  periblem  layer,  in  the 
production  of  buds,  but  only  an  anticlinal  in  the  dermat- 
ogen. 

For  an  example  of  the  flat  vegetative  cone  which  occurs 
in  most  phanerogams,  we  will  select  the  ornamental  shrub 
cultivated  in  most  gardens,  Evonymus  japonicus  (3). 
This  plant  may  be  had  at  any  time  of  the  year  and  its  buds 
readily  lend  themselves  to  section-making.  Prepare  a 
transection  in  order  to  2;et  a  view  of  the  cone  from  above. 
First  treat  the  section  with  potash  lye,  wash  with  water  and 
then  for  a  longer  time  with  acetic  acid.  With  a  low  mag- 
nification, we  recognize  the  cone  as  a  flat  knob  surrounded 
by  the  youngest  rudiments  of  leaves.  These  are  arranged 
in  a  two-limbed,  alternate  decussate  whorl.  Each  new 
pair  ©f  leaves  comes  forth  after  a  corresponding  growth  of 
the  vegetative  cone  in  the  spaces  between  the  two  preced- 


VEGETATIVE    CONE    OF    STEM. 


165 


ing  leaves,  Fig.  64,  A.  By  sufficient  magnification  the 
arrangement  of  the  cells  at  the  top  of  the  cone  is  easily 
made  out,  as  is  seen  in  Fig.  64,  B,     There  is  no  one  end- 


/        ^ 


Fig.  64.  End  of  the  stem  of  Evonyvius  japonicus.  A,  view  from  above  upon  the 
top.  X  12.  B,  view  of  the  apex  of  the  vegetative  cone.  X  240.  C,  median  longi- 
tudinal section  through  the  apex  of  the  stem.  X  28.  D,  median  longitudinal  sec- 
tion of  the  vegetative  cone.  X  240.  d,  dermatogen;  pr,  periblem;  pi,  plerome;/, 
beginning  of  the  leaf;  g,  beginning  of  a  bud ;  pf,  leaf  trace;  pc,  procambium  ring; 
m,  pith;  c,  rind. 

cell.  A  transection,  made  considerably  below  the  top, 
shows  a  rapid  diflerentiatiou  of  the  tissue,  into  funda- 
mental tissue,  "procambium,"  which  will  form  the  vascular 


166  VEGETATIVE    CONE    OF  "STEM. 

bundles,  antl  primary  rind.  The  procambium  zone  appears 
in  cross  section  as  a  rhomboid  figure,  with  somewhat  pro- 
jecting and  rounded  edges.  This  figure  is  alternatel}'  elon- 
gated in  the  direction  of  the  newly-entering  procambium 
cords.  The  procambium  consists  of  thin-walled,  narrow, 
radially-arranged  cells.  The  production  of  the  elements 
of  the  vascular  bundles  beij'ins  at  the  edges  of  the  fio;ure, 
protophlocm  elements  on  the  outer,  spiral  vessels  on  the 
inner  side  of  the  procambium  zone.  These  regions  of  the 
diiferentiation  of  the  elements  of  the  vascular  bundles 
are  not  distinctly  marked  off  from  the  rest  of  the  procam- 
bium tissue.  The  procambium  zone  opens  in  places  to  re- 
ceive the  entering  vascuhir  bundles  of  the  leaves.  In 
the  axils  of  the  young  leaves  one  may  see  the  beginnings 
of  the  axillary  buds.  The  median  longitudinal  section  is 
shown  with  slight  magnification  in  Fig.  64,  (J.  The  flat 
vegetative  cone,  the  leaf  l)egiunings  increasing  in  size,  the 
axillary  buds,  g,  the  differentiation  of  the  fundamental  tis- 
sue, w,  the  procambium  zone,^)c,  the  vascular  bundles  com- 
mon to  both  stem  and  leaves,  the  so-called  leaf-trace,^, 
and  the  primary  rind,  c,  are  recognized  at  a  glance.  Pith 
and  rind  have  a  large  number  of  ciystal  masses  of  calcium 
oxalate.  In  a  fresh  section  examined  in  water,  the  rind 
and  pith  appear  green,  wdiile  the  procambium  zone  is  quite 
clear.  Treat  with  potash  lye  and  acetic  acid  in  order  to 
follow  the  arrangement  of  the  cells  of  the  vegetative  cone. 
First,  we  come  to  the  single  layer  of  dermatogen  cells, 
Fig.  64,  jD,  d.  Next  these,  mantel-like  layers  of  the  peri- 
blem,  ^7',  and  then  the  plerome,  ^;?,  a  solid  central  C3'linder 
of  tissue  not  sharply  distinguished  throughout  from  the 
periblem.  The  vegetative  cone  appears  very  narrow  be- 
tween the  two  3'oungest  embryo  leaves,  but  one  may  often 
try  many  times  before  he  exactly  hits  the  first  beginnings 
of  the  leaf  and  makes  a  section  like  that  represented  in  Fig. 


VEGETATIVE   CONE    OF   STEM.  167 

64,  D.  Then  the  cone  appears  much  broader  and  the  his- 
togens  may  be  better  traced  out.  The  formation  of  the 
leaf  begins  with  the  periclinic  division  of  the  cells  in  the 
two  outer  layers  of  periblem,  f,  the  dermatogen  remain- 
ing a  single-celled  layer.  The  formation  of  the  axillary 
buds  takes  place  in  the  same  way,  in  the  axils  of  the  third 
youngest  pair  of  leaves  by  the  periclinal  division  of  the 
cells  of  the  hypodermal  layer.  In  general,  it  may  be  dem- 
onstrated that  the  dermatogen  furnishes  the  epidermis,  the 
periblem  the  rind,  and  the  plerome  the  pith  of  the  stem. 
It  is  less  certain  that  the  procambium  ring  arises  from  the 
plerome. 

That  the  formation  of  the  vascular  bundles  is  not  exclu- 
sively connected  with  the  plerome  follows  from  the  fact 
that  that  part  of  the  vascular  bundle  which  enters  the  leaf 
is  within  the  rind  and  is  therefore  produced  by  the  peri- 
blem and  that  the  entire  inner  tissue  of  the  leaf  with  its 
vascular  bundles  is  a  product  of  the  periblem. 

To  illustrate  the  growth  of  a  cryptogam  by  an  apical 
cell,  Ave  will  select  Equisetum  arvense  (4).  The  apical 
cell  is  easily  seen.  Use  a  growing  sprout,  taking  a  fresh 
one  or  one  preserved  in  alcohol.  Cut  off  a  piece  from 
the  top  about  10  mm.  long,  and  make  a  longitudinal 
section  between  the  fingers  as  already  described.  Find 
a  section  with  the  conical  tip  of  the  stem  intact.  Make  it 
transparent  by  the  addition  of  a  little  potash  lye.  Should 
this  be  so  strong  as  to  make  the  cell  wall  too  transparent 
and  therefore  unrecognizable,  weaken  the  solution  by  the 
addition  of  a  little  water.  In  fresh  sections  we  are  to 
avoid  the  use  of  all  dehydrating  substances  or  we  shall 
shrink  up  the  vegetative  cone.  Sections  from  alcohol  mate- 
rial may,  on  the  contrary,  be  examined  direct  in  glycerine, 
but  not  after  a  previous  soaking  out  in  water.  A  section 
treated  with  potash  may  advantageously  be  stained  with 


168 


VEGETATIVE    CONE    OF    STEM. 


a  very  dilute  solution  of  safranin.  The  staining  should 
be  very  slight  and  the  cell  walls  will  come  out  all  the  more 
distinctly.  We  get  the  best  results  where  we  treat  the 
section  for  a  short  time  with  concentrated  potash  solution, 
then  Avash  with  water  and  lay  it  for  two  hours  in  concen- 
trated acetic  acid.  Examine  in  water,  or,  better  still,  in 
dilute  acetic  acid,  or  a  concentrated  solution  of  potassium 
acetate.     A  permanent  preparation  may  be  made  with  the 


Fig.  65.  Longitudinal  section  of  the  vegetative  cone  of  a  sprout  of  Equisettim 
avense.  t,  apical  cell;  S',  youngest  segment;  S",  next  older  segment;/»,  principal 
wall;  tn,  halving  wall;  pr,  later  periclinal,«,  anticlinal  walls;/,  first,  f,  second,/", 
third  leaf  whorl;  g,  initial  cell  of  an  axillary  bud.  X  ^W- 

last  named  fluid.  Glycerine  shrinks  these  sections.  Ex- 
amine the  section  between  two  cover  glasses  as  already 
recommended  in  the  case  of  Hippuris. 

With  a  rightly-prepared  section  the  apical  cell  will  ap- 
pear in  the  form  of  a  triangular  inverted  pyramid  with  a 
convex  base,  Fig.  65,  t.  This  apical  cell  divides  by  walls 
which  are  parallel  to  the  existing  lateral  wall,  and  which 
follow  each  other  spirally  and  form  segments  arranged  in 


VEGETATIVE    CONE    OF    STEM. 


169 


three  exact  series.  These  segments  are  seen  in  profile  at 
Fig.  65,  S.  They  also  divide  in  a  definite  way  and  so 
the  plant  is  gradually  built  up.  At  some  distance  from 
the  apical  cell  a  wall  rises  from  the  vegetative  cone  which 
gi'ows  at  its  edge  by 
wedge-shaped  initial 
cells.  In  its  further  de- 
velopment the  edge  pro- 
trudes at  certain  places 
to  form  the  free  top  of 
the  leaf-whorl,  which 
grows  together  at  the 
base.  The  farther  we 
go  from  the  apical  cell, 
the  larger  becomes  the 
rudiments  of  the  leaf- 
whorl,  and  at  the  same 
time  the  difierentiation 
of  the  inner  tissue  of  the 
stem  goes  on,  princi- 
pally by  the  separation 
into  thick  small-celled 
low  nodes,  and  thinner 
long-celled  elongated 
internodes,  Fig.  66. 
The    wide-celled    pith 

,   ,        .  Fig.  66.    Median  longitudinal  section  througfi 

next  OeginS    to    aj^pear.  a  vegetative  sprout  of  Eqziisetum  arvense.  pv. 

The     first      rin»"     vessel  vegetative  cone  of  the  sprout;  ff,  initial  cell  of  a 

,                  °               ,  bud;   fir',  fir",  </'",  ^r'',  different  stages  of  devel- 

makeS  its  appearance  in  opment  ofsuch  buds ;r- and  »-'.tlie  beginnings  of 

the  fifth    highest    inter-  \™°'  «°  fhe  buds;  m,  differentiation  of  the 

i=>  original  pith;  vs,  entering  spiral  vessels;  n,  dif- 

node  in  theprocambium  ferentiatloa  of  the  node  diaphragm.  X  26. 

cord  on  the  outer  border  of  the  pith  and  from  here  may 
be  traced  into  the  beginniusr  of  the  next  hiofher  leaf-whorl. 
Each  single  vascular  bundle  is  common  to  both  the  stem 


170  VEGETATIVE    CONE    OF    STEM. 

and  the  leaf  and  may  hence  ])e  called  a  leaf-trace.  As 
many  vascular  bundles  run  downward  in  each  internode 
as  there  are  leaves  in  the  whorl.  The  separate  leaf-traces 
first  become  connected  by  lateral  branches  somewhere 
about  the  lower  half  of  the  seventh  internode,  thus  form- 
in<r  closed  vascular  bundles.  Near  the  tenth  internode 
the  pith  begins  to  become  hollow,  by  the  weakening  and 
separation  of  the  cells.  In  the  nodes,  on  the  contrary, 
the  cells  of  the  pith  have  a  corresponding  increase  and 
continue  coherent.  The  lateral  buds  arise  from  single  cells 
in  the  axils  of  the  leaf-whorls.  They  stand  in  whorls 
and  alternate  with  the  free  points  of  the  leaves  in  the  leaf 
whorls,  the  tissue  of  which,  at  the  base,  they  perforate  in 
cro^vins:  out.  The  lonoitudinal  section  shows  the  some- 
what  larger  rudimentary  bud  growing  in  the  tissue  of  the 
thick  leaf-whorl  which  lies  upon  the  surface  of  the  stem. 
Somewhere  near  the  seventh  node  the  buds  are  so  far  de- 
veloped as  to  possess  several  rudimentary  leaf-whorls  which 
may  be  advantageously  used  for  studying  the  apical  cell. 
Among  the  cryptogams  only  the  Equisetoe,  and  the  Opld- 
oglossoe  bive  collateral  vascular  bundles.  The  bundles 
are  arranged  in  a  simple  ring  about  the  hollow  pith.  In 
the  wood  part  of  each  bundle  is  an  intercellular  passage, 
the  carina!  cavity.  The  thin-walled  bast  portion  is  in- 
closed on  the  sides  by  the  ring  and  reticulated  vessels  of 
the  wood  part.  An  endoderm  surrounds  the  whole  vas- 
cular tissue  body.  In  the  broad  rind,  alternating  with  the 
vascular  bundles  are  the  wide  intercellular  passages,  the 
vascular  cavities.  The  number  of  vascular  bundles  ex- 
actly corresponds  with  that  of  the  leaf  tips  in  the  whorl 
next  al)ove.  In  order  to  observe  the  course  of  the  vascu- 
lar bundles,  make  a  whole  series  of  transections  down 
through  the  lower  part  of  the  internode,  through  the  node 
and  into  the  next  lower  internode.     For  this  purpose  we 


VEGETATIVE    CONE    OF   STEM.  171 

may  use  alcohol  or  fresh  material,  but  should  take  the 
youngest  possible  pai-t  of  the  stem,  as  the  older  parts  are 
silicified  and  soon  dull  the  knife.  Use  the  microtome  for 
this.  The  sections  should  be  arranged  in  their  order  on 
the  slide  and  be  made  transparent  with  potash  lye.  By 
an  exact  comparison  of  these  successive  sections  we  shall 
learn  that  each  of  the  vascular  bundles,  as  it  descends  from 
the  internode  above,  splits  into  two  branches  in  the  node 
and  each  of  the  branches  unites  with  a  neio^hborinsr  vascu- 
lar  bundle  coming  down  into  the  node  from  the  leaf-whorl, 
thus  forming  a  new  bundle.  If  the  bundles  of  the  lateral 
buds  are  already  developed  they  will  complicate  the  ar- 
rangement somewhat.  Each  lateral  bud  is  connected  with 
the  vascular  system  of  the  mother  axis  by  two  vascular 
bundles,  and  indeed  with  each  bundle  to  the  two  forking 
branches  of  a  bundle  from  the  next  higher  internode  of  the 
stem,  immediately  after  it  separates  into  its  two  branches. 
The  lateral  buds  alternate  with  the  vascular  bundles  of  the 
leaf- whorls  which  cover  them,  and  correspond  in  their 
position  to  the  vascular  bundles  of  the  next  higher  and 
next  lower  whorls.  It  follows  from  our  observations  that 
the  vascular  system  is  common  to  the  whole  plant  and  is 
formed  of  leaf-traces  which  divide  at  their  base  within 
the  node  and  by  means  of  their  foi-king  branches  pass  in- 
to other  bundles  entering  the  node  from  aI)ove  and  below. 
As  this  is  the  method  of  the  formation  of  the  vascular 
system  generally  in  vascular  plants,  we  shall  limit  our 
studies  of  the  same  to  this  simplest  example.  In  the  in- 
vestigation of  a  complicated  case  it  is  necessary  to  arrange 
the  successive  sections  on  the  slide  in  the  same  position 
for  purposes  of  comparison.  This  may  most  easily  be 
done  by  cutting  a  longitudinal  slit  down  the  side  of  the 
specimen  which,  of  course,  in  each  section,  Avill  indicate 
the  corresponding  side.     It  is  often  necessary  to  draw  the 


172  VEGETATIVE   CONE   OF   STEM. 

section,  in  order  to  be  able  to  demonstrate  the  shifting  of 
a  single  bundle  with  certainty.  Tangential,  longitudinal 
sections  made  transparent  with  potash  may,  in  many  cases, 
lay  bare  in  a  single  section,  the  whole  course  of  a  vas- 
cular bundle. 

Notes. 

(1)  Sanio,  Bot.  Zeitung,  1864,  p.  223,  Anna.  .  .,  1865,  p.  184;  de 
Bary,  Vergl.  Anat.,  p.  9;  L.  Kny,  Wandtafln,  in  Abth.,  p.  99. 

(2)  Sachs,  Arbeiten  des  Bot.  Inst,  in  Wiirzbui-g,  Bd.  ii,  p.  46,  u.  185. 

(3)  Haustein,  die  Sclieitelzellgrnppe  im  Vegetationspunct  d.  Phan- 
erogamen,  p.  9 ;  Warming,  Rech.  s.  1.  ramif .  d.  Phaner. 

(4)  See  Cramer,  Pflanzenphys.,  Unters,  v.  Nägeli,  Heft  3,  p.  21; 
Keess,  Jahrb.  f.  wiss.  Bot.,  Bd.  vi,  p.  209;  Sachs'  Lehrb.,  iv  Aufl., 
p.  393  und  Goebel,  Grudzgüge,  p.  291 ;  de  Bary,  Vergl.  Anat.  p.  20. 


LESSON  XVII. 
Vegetative  Cone  of  the  Eoot. 

We  shall  now  study  the  vegetative  coue  of  the  root  (1) , 
beginning  with  the  angiosperms,  and  taking  our  speci- 
men from  the  relatively  easy  Graminacece.  It  furnishes 
but  one  of  the  many  possible  types  of  the  root  growth  of 
the  angiosperms,  but  it  is  one  quite  widely  distributed  and 
very  instructive  in  respect  to  the  process  in  question. 
Take  a  plant,  the  common  barley,  Hordeum  vulgare,  grown 
in  a  flower  pot.  Tilt  the  pot  so  as  to  find  the  free  ends 
of  the  roots  in  the  outside  of  the  soil,  and  make  the  in- 
vestigation with  fresh  material.  Make  a  transection  of  an 
old  part  of  the  root.  We  shall  find  a  large  vessel  in  the 
middle  of  the  axile  fibro-vascular  bundle  cylinder,  and 
arranged  about  it  some  eight  vascular  rays  alternating  with 
an  equal  number  of  bast  parts.  The  vascular  rays  extend 
here  to  the  endoderm,  therefore  interrupting  the  pericam- 
bium.  The  endoderm  shows  more  or  less  distinctly  the 
dark  radial  shadows.     Then  follows  a  pretty  stout  rind. 

Make  an  exactly  median  longitudinal  section  of  the  end 
of  the  root,  between  thumb  and  finger  as  in  the  other  case, 
and  examine  without  reagents.  Before  all,  make  sure 
that  the  body  of  the  root  is  seen  sharply  distinct  from  the 
root-cap.  A  line  may  l)e  traced  which  follows  the  outer 
surface  of  the  epidermis  over  the  apex  between  the  body 
of  the  root  and  the  root-cap.  See  Fig.  67.  The  dernia- 
togen  does  not,  as  such,  extend  over  the  top,  but  rather  it, 
d,  and  the  periblem,^?',  come  to  this  point  at  the  top  in 
common  initial  cells.  In  the  illustration  beyond,  only  one 
such  common  cell  occurs  ;  there  may  be  several.    The  der- 

(173) 


174 


VEGETATIVE    CONE   OF   ROOT. 


matogen  may  be  traced  to  these  initial  cells  ;  the  periblem, 
a  single  layer  thick,  also  touches  it.  The  plerome  comes 
to  a  point  under  this  cap  in  its  own  initial  cells.     On  the 


Fig.  67.  Median  long-ittulinal  section  of  the  end  of  a  root  of  Hordeum  vulgare, 
k,  calyptrogen  ;  c,  tliickeneil  outer  wiill  of  epidermis ;  en.  endoderm  ;  i,  intercellu- 
lar passage  filled  with  air;  a,  a  cell  series  wliicli  will  form  the  central  vessel;  r, 
disengaged  cells  of  the  root  cap.  X  180. 

line  which  separates  the  body  of  the  root  from  the  root- 
cap,  toward  the  outside,  are  the  initial  cells  of  the  cap, 
forming  a  layer  of  flat  cells  and  called  calyptrogen,  k. 


VEGETATIVE  CONE  OF  EOOT.  175 

These  cells,  in  accordance  with  their  origin,  are  arranged 
in  rows,  first  flat  and  then  attaining  considerable  height 
afterwards;  at  the  top  of  the  cap  rounded  out  and  finally 
separated  from  each  other  and  become  disorganized,  r.  A 
peculiarity  of  the  Graminacece  is  that  the  dermatogen  is 
strongly  thickened  on  the  outside,  c.  It  is  sparkling  white 
and  becomes  thicker  the  lono^er  it  lies  in  the  water.  On 
the  lateral  borders  of  the  cells  may  be  seen  strongly  refrac- 
tive strips  continued  more  or  less  deeply  into  the  thickened 
outer  wall.  They  are  those  of  the  primary  walls  of  the  cell 
and  indeed  reach  further  into  the  thickened  walls  the 
deeper  they  are.  These  walls  show  distinct  lamination. 
The  periblem  has  rapidly  increased  its  cell  layers  by  peri- 
clinal  divisions.  Between  the  inner  ones  air-filled  inter- 
cellular passages  very  soon  begin  to  enter,  designated  in 
the  illustration  by  dark  lines.  Fig.  67,  at  ^.  The  peri- 
blem produces  the  rind,  the  innermost  layer  of  which  be- 
comes the  endoderm.  The  plerome  ends  in  a  cone-shaped 
group  of  initial  cells,  two  such  being  discernible  in  the 
illustration.  The  plerome  foruis  the  axile  fibro-vascular 
bundle  cylinder.  The  differentiation  of  the  large  central 
vessels  in  the  bundle  may  be  traced  quite  up  to  the  initial 
group,  the  cells  which  shall  form  them  being  indicated  by 
their  greater  breadth,  a.  Those  which  shall  form  the 
smaller  vessels  will  be  first  distinguishable  much  later. 

The  roots  of  the  gymnosperms  show  in  many  connec- 
tions a  peculiar  articulation  in  the  meristem  of  the  vege- 
tative cone.  Let  us  study  Tliuia  occideiitalis.  A  transection 
throuo^h  a  fuU-orown  root  resembles  that  of  the  Taxus  bac- 
cata  only  that  the  root  of  the  Thuia  is  quadrilaterally 
built.  A  median  longitudinal  section  through  the  end  of 
the  root  shows  a  well-defined  plerome  cylinder  which  ends 
in  a  few  initial  cells  and  is  surrounded  by  a  mantle  of  per- 


176  VEGETATIVE  CONE  OF  ROOT. 

iblem  twelve  to  fourteen  layers  of  cells  thick.  The  latter 
continues  over  the  end  of  the  root  and  forms  there  its 
inner  series  of  eight  to  ten  colored  initial  layers  while  the 
outer  series  passes  over  into  irregularly-arranged  relatively- 
large  cells.  These  large  cells  extend  to  the  top  of  the 
root-cap  where  they  finally  separate  and  fall  away.  The 
root-cap  of  Thuia,  as  of  the  gymnosperms  generally,  con- 
sists of  the  outer  elements  of  the  periblem,  dermatogen  and 
calyptrogen  both  failing.  The  initial  layers  of  the  peri- 
blem divide  by  both  periclinal  and  anticlinal  walls.  The 
periclinal  division  increases  the  number  of  periblem  layers 
and  supply  from  within  the  cells  thrown  off  from  the  pe- 
riphery. The  anticlinal  divisions  increase  the  number  of 
cells  in  the  single  layers  and  provide  principally  for  the 
building  up  of  the  rind.  The  periclinal  dividing,  in  the 
initial  laj^er  of  the  apex,  produces  this  result :  that  the  cell 
series  of  the  rind  when  followed  out  to  the  point  seems  to 
be  doubled.  The  central  straight  anticlinal  cell  row  in  the 
periblem  of  the  apex  of  the  root  is  much  more  distinctly 
conspicuous  than  the  neighboring  cells.  It  forms  the  "per- 
iblem column"  which  loses  itself  in  the  brown  cells  of  the 
root-cap.  This  column  appears  clearer,  its  cells  immedi- 
ately touching  each  other,  while  they  laterally  form  adja- 
cent air-filled,  intercellular  spaces.  These  cells  are  also 
distinguished  by  their  rich  starch  contents.  The  roots  of 
the  Thuia  possess  no  epidermis,  the  surface  of  the  root 
being  covered  with  the  outer  layer  of  the  periblem.  If 
we  follow  this  layer  in  the  direction  of  the  end  of  the  root, 
we  shall  soon  find  it  extending  under  another  which  now, 
for  some  distance,  constitutes  the  outer  surface.  This 
outermost  living  cell  layer  is  protected  on  its  outer  surface 
hy  the  collapsed  and  browned  cell  walls  of  a  dead  cell 
layer.     The  roots  of  Thuia  and  of  the  gymnosperms  gen- 


VEGETATIVE  CONE  OF  THUIA  ROOT. 


177 


erally  have  no  root  hairs.  Fig.  68  shows  a  slightly  ma»"- 
nified  image  of  a  longitudinal  section  through  the  end  of 
the  root  in  which  the  various  parts  can  be  easilj'^  made  out. 
We  see  first  the  brown  cell-sheath,  x,  then  the  periblem, 
^r,  Avhich  extends  over  the  apex  of  the  root  and  whose  outer 
layer  there  forms  the  root-cap  ;  finally 
the  plerome,  pi,  whose  upper  termi- 
nation is  not  clearly  seen  with  so  small 
a  magnification.  One  may  easily  think 
the  upper  part  of  the  plerome  to  be 
more  bulky  than  it  really  is,  because 
the  innermost  layer  of  the  periblem 
borders  on  the  plerome  without  an  iu- 
terceUular  space  and  appears  as  clear 
as  the  plerome  cylinder  itself.  In  the 
oldest  part  of  the  section  the  plerome 
cylinder  is  covered  by  a  la^'er  of  red 
cells,  which  correspond,  as  a  compar- 
ison with  the  transection  shows,  with 
the  endoderm  cells  filled  with  red  cell- 
sap.  At  some  distance  from  the  end 
of  the  root  these  cells  are  still  unrec- 
ognizable. Vessels,  s,  are  formed  in 
the  older  parts  of  the  plerome  cylinder. 
The  apex  of  the  periblem  is  occupied 
with  the  conspicuous  periblem  column, 

-  bium;  p<,  plerome;  e,  en- 

c,  against  which,  laterally,  the  layers  of  dodeim;  s.  spiral  vessels,- 

periblem  abut.     The  latter  extend  nei-  r;^^',!  x  26."'"'""'  '' 

ther  to  the  plerome  nor  to  the  outer 

surface  of  the  root,  which  last  is  covered  by  large  brown 

cells. 

We  will  make  use  of  a  coniferous  plant  in  studying  the 
methods  of  root-branching.     We  observe  in  the  roots  of 
12 


Fig.  GS.  Longitudinal 
section  of  the  end  of  a 
root  of  Thuia  occidentalis. 
X,  outer  brown  layer  of 
cast-off  cells  ;/>r,  pericam- 


178 


VEGETATIVE    CONE    OF   ROOTS. 


Thuia  occidentalis  that  they  bear  lateral  rootlets  in  four, 
and  at  last  in  three,  straight  rows.  By  making  a  section 
of  the  root  we  find  that  these  rows  of  rootlets  correspond 
first  to  the  four-  and  then  to  three-sided  vascular  bundle 
cylinders  in  the  roots.     By  making  a  section  through  the 


Fig.  C9.  Median  longitudinal  section  of  a  root  of  Pteris  critica.  t,  apical  cell; 
k,  initial  cell  of  cap;  Jc",  outer  cap;  c,  e,  r, p,  cambium,  epidermis,  rind  and  peri- 
cambium  walls  respectively.  X  '^lO. 

root  at  the  j)oint  of  insertion  of  the  rootlet  we  find  that  the 
rootlets  stand  before  a  wood  part  of  the  cylinder,  and  since 
these  wood  parts  run  straight  along  the  axis  of  the  vascu- 
lar cylinder  the  observed  arrangement  of  the  lateral  root- 
lets  is  explained.      We   will    now    undertake   to   learn 


VEGETATIVE  CONE  OF  FERN  ROOTS.       179 

somethins:  of  the  veofetative  cone  of  a  root  which  otows 
by  means  of  an  apical  cell  (3)  There  is  no  such  variety 
of  forms  in  the  roots  as  in  the  stems  ^vhich  grow  b}'  this 
means.  The  three-sided  pyramidal  apical  cell  occurs,  how- 
ever, and  the  articulation  by  the  formation  of  segments 
remains  constant.  Take  the  root  of  ^i^enscnV2ca,  Fisf.  69. 
It  would  be  quite  as  well  to  select  another  form.  By  tilt- 
ing the  flower  pot  we  shall  easily  obtain,  uninjured,  the 
end  of  the  root.  The  roots  of  Pteris  critical  as  of  ferns 
generali}^  are  bilaterally  constructed,  flat  bast  parts  alter- 
nating with  the  wood  ;  the  pericambium  consists  of  one 
layer,  the  endoderm  is  flat  and  the  inner  part  much  thick- 
ened. Prepare  a  median  longitudinal  section  as  already 
directed.  It  is  not  difficult  to  bring  the  apical  cell  here 
into  view.  It  does  not  take  in  the  apex  of  the  root,  but 
is  covered  with  the  root-cap.  The  apical  cell.  Fig.  69,  t, 
like  that  of  the  stem  of  Equisetum,  has  the  form  of  a  tri- 
angular pyramid  whose  convex  base  is  turned  toward  the 
root-cap,  while  the  apex  is  sunk  in  the  body  of  the  root. 
The  divisions  succeed  each  other  as  in  the  Equisetum  par- 
allel to  the  lateral  surfaces,  but  besides  this  there  will  oc- 
cur from  time  to  time,  mostly  after  every  three  of  the 
described  divisions,  the  formation  of  a  wall  in  the  direc- 
tion of  the  surfiice  of  the  base.  The  cell  produced  by  this 
has  nearly  the  form  of  a  segment  of  a  globe.  This  cell,  a-, 
is  an  initial  cell  for  the  root-cap,  forms  a  cap-like  cell 
layer  and  is  the  origin  of  the  root-cap.  It  divides  into 
halves  by  a  wall  perpendicular  to  the  under  surface.  These 
halves  repeat  the  division,  thus  forming  four  four-sided 
cells.  Qy  a  constant  repetition  of  this  process  of  division, 
that  is  by  walls  perpendicular  to  the  basal  wall,  an  old  cap, 
Ä;",  will  consist  of  a  large  number  of  cells.  The  cells  of 
old  caps  are  filled  with  starch  grains.     They  become  grad- 


180  VEGETATIVE  CONE  OF  EOOTS. 

ually  disorganized,  while  the  apical  cell  constantly  pro- 
duces new  initial  cells.  The  outer  walls  of  the,  for  the 
time  being,  outer  layer  of  cap-cells  become  much  thickened. 
The  division  walls  formed  parallel  to  the  sides  of  the  api- 
cal cell  follow,  as  in  the  stem  of  the  Equisetum,  the  direc- 
tion of  a  spiral. 

Notes. 

(1)  Sachs,  Lehrb.,  iv  Auflag,,  p.  166;  v.  Janczewski,  Ann.  d.  sc. 
nat.  Bot.,  V  Ser.,  T.  xx,  1873,  p.  162ff. ;  Treub,  Musee  bot.  de  Leide, 
T.  II,  1876;  de  Bary,  vergl.  Anat.,  1877,  p.  10. 

(2)  Strasburger,  Coniferen  und  Gnetaceen,  p.  .^40 ;  de  Bary,  vergl, 
Anat.,  p.  14.     See  there  also  the  further  literature. 

(3)  Nägeli  u.  Leitgeb,  iuBeitr.  zur  wiss.  Bot.,  4  Heft,  1868,  p.  74ff. 


LESSON  XVIII. 

Histology  of  the  Mosses. 

Heretofore  we  have  studied  the  structure  of  the  stem 
and  leaves  in  the  vascular  plants  only.  We  will  now  turn 
to  the  small  stems  and  leaves  of  the  mosses,  which  are 
quite  without  vessels  (1).  We  will  begin  with  Mnium 
undulatum,  a  relatively  complicated  case,  in  which  the  dif- 
ferentiation of  tissue  is  quite  well  advanced.  Make  first  a 
delicate  transection  through  the  stem.  In  the  middle  of  the 
stem  is  an  axilhiry  cylinder  formed  of  narrow  thin-walled 
cells.  We  may  consider  this  cylinder  as  the  simplest 
"conducting  bundle."  Its  cells  have  no  living  contents, 
but  contain  water  only.  They  are  distinguished  from  the 
surrounding  cells  by  the  yellowish-brown  color  of  their 
walls.  Upon  the  conducting  bundle  abut  the  wide  cells 
of  the  rind,  which  are  much  larger,  with  greenish-yellow 
walls,  and  living,  chlorophyll-containing  contents.  They 
increase  somewhat  in  width  towards  the  outside  but  at  the 
periphery  become  suddenly  narrow  and  thick-walled,  and 
pass  over  without  definite  demarcation  into  the  epidermis, 
which  consists  of  one  or  two  layers  of  much  thickened  cells. 
In  two  or  three  places  the  outer  layer  of  cells  of  the  stem 
is  continued  into  a  cell-plate  of  a  single  layer,  Avhich  cor- 
responds to  the  downward  running  leaf-wing  on  the  stem. 
A  section  made  below  the  leaves  in  the  stouter  brown  part 
of  the  stem  shows  the  walls  of  the  peripheral  cell  layer 
colored  a  dark  brown.  From  single  cells  of  the  surface 
grow  long,  brown- walled,  many-times-branched  cell  fibres, 
designated  root-hairs  or  rhizoids,  which  do  duty  as  roots. 
These  rhizoids  arc  distinguished  by  oblique  division  walls, 

(181) 


182  HISTOLOGY    OF   MOSSES. 

and  are  hence  an  exception  to  the  general  rule  which  would 
demand  an  exactly  transverse  wall.  Under  many  such  di- 
vision walls  and  indeed  beneath  their  elevated  edge  spring 
wider  spreading  lateral  l)ranches.  Only  the  growing  ends 
of  the  rhizoids  have  colorless  walls. 

These  root  fibres  exhibit  the  greatest  resemblance  in  re- 
spect to  branching,  and  the  inclined  division  walls,  to  the 
primary  growth,  the  so-called  protonema,  of  the  typical 
mass,  which  is  first  developed  from  a  sprouting  spore. 
Still  these  branches,  when  they  do  not  penetrate  the  soil, 
are  colorless  and  bear  chlorophyll  grains.  The  leaf  buds 
which  develop  into  moss  stems  are  lateral  branches  of  this 
protonema.  The  near  relation  of  rhizoids  and  protonema 
is  seen  also  from  the  circumstance  that  the  rhizoids  damp- 
ened and  set  out  in  the  light  can  produce  protonema  which 
will  give  rise  to  numerous  new  plants.  It  is  only  nec- 
essary to  lay  a  tuft  of  Milium  bottom  side  up  and  keep  it 
damp  in  order  to  luoduce  a  rich  green  protonema  mass 
from  the  rhizoids,  which  resembles  terrestrial  VaucJieria 
tufts  in  its  general  appearance. 

If  the  section  should  be  made  through  some  point  in  the 
stem  of  the  Mnium  which  had  been  injured  we  shall  not 
find  the  injury  repaired  by  being  closed  up  with  a  layer  of 
cork,  for  the  cryptogams  with  the  exception  of  Botry- 
chium  cannot  form  cork,  but  the  walls  of  the  adjacent  cells 
will  be  thickened  and  browned  so  that  they  will,  with  the 
exception  of  their  greater  interior  diameter,  resemble  the 
other  cells  of  the  outer  surface. 

The  transection  will  show  near  the  surface  of  the  stem 
single  small  strings  of  thin-walled  cells  which  agree  in 
color  and  in  their  function  as  carriers  of  water,  Avith  the 
cells  of  the  central  cylinder.  These  are  the  conducting 
bundles  belonging  to  the  leaves  and  end  blindly  in  the 
rind  of  the  stem.     In  PolytricJium,  however,  they  extend 


HISTOLOGY   OF   MOSSES.  183 

further  inward  and  connect  themselves  with  the  central 
conducting  bundle  of  the  stem.  Put  a  leaf  in  a  drop  of 
water  on  the  slide.  We  shall  find  it  to  consist  of  a  single 
layer  of  cells  with  a  middle  nerve  of  several  layers,  the 
latter  ending  in  a  terminal  tooth  which  consists  of  a  num- 
ber of  rhombic  cells.  The  cells  of  the  mid-rib  are  much 
elongated,  and  the  outer  ones  contain  chlorophyll  grains. 
The  cells  of  the  leaf  are  polygonal  and  also  contain  chloro- 
phyll. The  bandlike  hem  around  the  edge  of  the  leaf  is 
formed  of  elongated,  much-thickened  cells.  At  nearly 
regular  intervals  on  the  outer  edge  are  sharply  pointed 
teeth  one  or  two  cells  long.  We  may  get  sections  of  the 
leaf  at  the  same  time  that  we  make  sections  of  the  stem. 
But  if  we  wish  to  make  sections  of  the  leaf  separately,  we 
may  fasten  a  number  of  them  together  with  glycerine  gum, 
and  without  waiting  for  the  gum  to  dry,  make  sections  of 
the  whole  between  pieces  of  elder  pith.  Then  lay  the 
sections  in  water  and  the  gum  will  be  dissolved.  This 
method  is  recommended  for  very  thin  flat  sections.  We 
see  from  one  section  that  the  leaf  consists  of  one  layer 
of  cells  and  that  the  cells  of  the  leaf-hem  are  very  much 
thickened.  The  nerve  projects  more  on  the  back  than  on 
the  front  of  the  leaf,  and  in  the  middle  of  it  somewhat 
nearer  the  under  side  lies  a  string  of  thin  walled  cells,  the 
conducting  bundle  which  we  before  saw  in  the  rind  of  the 
stem.  This  string  is  protected  behind  by  some  much 
thickened  narrow  cells.  The  image  reminds  us  not  a  little 
of  certain  much  reduced  monocotyledonous  vascular  bun- 
dles, which  consist  of  a  few  bast  elements  and  a  thin  layer 
of  sclerenchyma  cells. 

If  the  stem  of  a  wilted  plant  be  put  in  the  water  the 
plant  will  remain  wilted,  but  if  the  leaves  be  put  in  the 
water  the  plant  will  rapidly  become  turgescent.  The  leaves, 
therefore,  are  the  principal  absorbents  of  water,  which  fact 


184  HISTOLOGY   OF   MOSSES. 

renders  a  direct  connection  of  the  condncting  bundle  of 
the  leaf  Avith  that  of  the  stem  quite  superfluous. 

The  turf  moss  offers  certain  striking  peculiarities  which 
we  will  now  consider.  Make  a  transection  of  the  stem  of 
/Sphagnum  aculifolium.  The  section  shows  us  a  wide  cen- 
tral cylinder  consisting  of  wide  somewhat  collenchyma- 
tously  thickened  cells  ;  towards  the  periphery  the  cells 
become  gradually  narrower,  and  in  the  outermost  layer  are 
colored  a  yellow  brown.  There  is  no  specialized  conduct- 
ing bundle  in  the  interior  of  the  cA'linder,  which  is  inclosed 
by  an  outer  rind  of  large  cells  three  layers  thick.  These 
cells  lie  next  the  narrow  yellow-brown  cells  of  the  inner 
cylinder.  They  are  distinguished  by  their  large  round  or 
oval  orifices  and  their  delicate  spiral  bands.  The  open- 
ings in  the  walls  really  connect  the  cell  cavities  of  adjacent 
cells  as  may  be  seen  when  the  section  touches  one  of  them. 
One  often  sees  the  mycelium  of  a  fungus  passing  through 
these  openings  from  cell  tocell  without  hindrance.  These 
porous  cells  of  the  outer  walls  of  Sphagnum  contain  only 
Avater  or  air  and  have  no  living  contents.  They  serve  the 
plant  only  as  capillary  apparatus  by  which  the  water  is  con- 
veyed to  the  place  where  it  is  to  be  used.  The  plant  has 
no  cutinized  cell  walls  ;  concentrated  sulphuric  acid  dis- 
solves the  whole  tissue,  but  the  middle  lamella  and  the 
pores  of  the  yellow-brown  outer  cells  of  the  central  cylin- 
der resist  the  action  of  the  acid  longest. 

The  extended  leaf  is  ovate,  bordered,  one  layer  of  cells 
thick,  and  consists,  as  a  superficial  view  will  teach,  of 
two  kinds  of  elements  :  one,  of  living  cells  with  proto- 
plasm nucleus  and  chlorophyll  grains  ;  the  other  of  dead 
cells  filled  with  water  or  air,  and  furnished  with  rings  or 
spiral  bands  and  openings  between  the  cell  cavities.  The 
reason  why  dead  cells  used  to  carry  water  or  air  so  often 
have  their  cell  walls  strengthened  with  spiral  bands,  rings 


HISTOLOGY   OF   LIVERWORTS.  185 

or  reticulations  is  because  they  have  lost  their  turgiclity 
and  must  have  some  such  mechanical  support  for  their 
walls  in  order  not  to  collapse  or  become  compressed.  The 
green  cells  of  the  leaf-blade  are  all  connected  together 
and  form  a  network  with  elegant,  bent  walls  whose  meshes 
are  occupied  each  by  an  empty  cell.  The  green  cells  serve 
for  the  assimilation  of  carbon,  the  empty  ones,  like  those 
of  the  stem,  for  conducting  water.  The  outer  edge  of  the 
leaf  is  occupied  by  slender  green  cells,  and  at  the  conclu- 
sion of  these,  by  a  slender  border  of  cells,  a  single  layer 
thick,  bearing  watery  contents,  slightly  thickened  on  the 
outside  and  somewhat  collapsed.  Only  the  ends  of  these 
cells  seem  much  thicker  and  project  a  little. 

There  is  no  nerve  in  the  leaves  as  there  is  no  conduct- 
ing bundle  m  the  stem.  The  plant  is  therefore  much  more 
simply  constructed  than  the  Mnium  in  this  respect,  but 
more  complicated,  on  the  other  hand,  in  being  provided 
with  a  special  capiUary  apparatus. 

The  well-known  MarcJiantia  polymorpha  (2)  presents  a 
pretty  complicated  structure.  The  lack  of  a  cormophytic 
articulation  does  not  necessarily  imply  a  simple  anatomi- 
cal structure.  The  thaUus  is  hard  and  leathery.  It 
branches  by  the  forking  of  the  growing  point  which  lies 
at  the  bottom  of  the  apical  sinus.  If  a  sprout  has  but  re- 
cently forked,  the  middle  of  the  anterior  indentation  will 
be  occupied  by  a  thallus-lobe,  at  the  two  sides  of  which 
lie  the  apical  sinuses.  In  the  middle  of  each  ]ol)e  on  the 
under  side,  an  indistinctly-outlined  mid-rib  projects. 
Stripes  run  out  diagonally  forward  from  these,  bending 
toward  the  edge  of  the  frond.  At  some  distance  from  the 
end,  fine  rhizoitls  spring  from  the  middle  of  the  thallus 
and  serve  to  fix  it  to  the  ground.  By  examining  the  luider 
side  of  the  plant,  inider  the  simplex,  we  can  demonstrate, 
by  the  help  of  a  needle,  the  presence  of  scales  springing 


186 


HISTOLOGY   OF   LIVERWORTS. 


from  the  surface  of  the  thalhis.  There  are  three  distinct 
forms  of  these  ventral  scales :  those  which  grow  on  the 
edge  of  the  frond,  those  which  grow  in  the  middle  and 
those  which  are  inserted  between.  The  second  and  third 
sorts  of  scales  give  the  stripes  which  we  have  observed 
with  the  naked  eye.  Viewed  with  a  lens,  the  back  side  of 
the  frond  appears  to  be  divided  into  small  rhomboid  fields, 
the  boundaries  of  which  are  dark  green,  the  fields  them- 
selves more  grayish.  In  the  middle  of  each  is  a  minute 
opening.     Examining  a  section  made  parallel  to  the  back 


Fig.  70.    Marchantia  polymoiyha.  A,  stoma  seenffom  above;  B,  in  transection. 

side  of  the  thallus  with  a  higher  magnification  and  we  see 
that  the  outer  cells  are  polygonal,  closely  connected  and 
contain  numerous  large  chlorophyll  grains.  The  round 
opening  in  the  middle  of  each  field  is  bordered  by,  at  most, 
four  slender,  bent,  sickle-form,  chlorophyll-free  cells, 
Fig.  70,  A.  When  the  section  reaches  deeper,  air  collects 
under  the  surface  of  the  fields.  Into  these  air  cavities, 
"air  chambers,"  chlorophyll-containing  cell  fibres  project. 
The  lateral  walls  of  the  air  chambers  are  constructed  of 
closely  connected  cells,  one  to  several  layers  thick  and 
contain   chlorophyll.     In  single  cells  of  the  surface  are 


HISTOLOGY   OF   LIVERWORTS.  187 

seen  certain  bodies  characterized  by  their  strong  refractive 
power,  their  irregular  outline  and  cluster-like  form.  In 
young  shoots  these  bodies  are  faintly  brown,  in  older, 
brown,  containiug  mostly  only  fatty  oil  and  form  the  so- 
called  "oil  bodies"  of  the  liverworts  (3).  A  superficial 
section  made  from  the  under  side  of  the  thallus  shows  no 
fields,  while  the  cells  are  elongated  and  coutain  less  chlo- 
rophyll than  those  of  the  upper  side.  The  rhizoids  ex- 
hibit a  double  structure.  They  are  slender  and  provided 
with  conical  projectious,  or  thicker  and  without  these. 

The  former  take  their  rise  from  that  portion  of  the  frond 
covered  by  the  middle  and  the  intermediate  scales  or  by 
the  former  only.  They  lie  close  to  the  frond,  quite  up  to 
the  middle  nerve,  are  covered  with  the  scales  and  serve 
to  stiffen  the  thallus.  The  common  rhizoids  arise  princi- 
pally from  the  middle  nerve  aud  turn  with  a  uniformly 
acute  angle  toward  the  substratum  upon  which  they  fasten 
the  thallus.  At  their  points  they  are  often  lobed  and  at 
their  base  colored  purple.  All  ventral  scales  consist  of 
one  layer,  the  middle  of  living,  the  other  two  of  dead  cells. 

A  cross-section  of  the  thallus  shows  it  to  consist  on  the 
upper  side  first  of  a  zone  of  chlorophyll-containing  tissue, 
then  within,  of  wide  cells  almost  destitute  of  chlorophyll. 
On  the  under  side,  the  last  two  layers  of  cells  are  again 
narrow,  flat,  and  rich  in  chlorophyll,  forming  the  so-called 
"ventral  rind  layer."  Oil  bodies  are  scattered  through  the 
whole  tissue.  Muciperous  cells,  which  are  distinguished 
by  their  size  and  refractive  qualities,  are  but  poorly  devel- 
oped in  the  Marchantia,  but  much  more  richly  in  other 
related  genera.  Looking  now  at  the  upper  portion  of 
the  transection,  we  first  see  a  simple  layer  of  flat  cells 
which  over  the  air  chamber  are  set  free  on  the  walls  which 
form  the  side?*  of  the  chamber.  In  the  middle  of  the  free 
outer  wall  is  the  breathing  place,  which,  as  it  now  shows 


188  HlSTOLOGr   OF   LIVERWORTS. 

itself,  is  inclosed  by  several,  from  four  to  eight  stories  of 
cells  (4).  See  Fig.  70,  B.  The  opening  is  narrowed 
above  and  below.  The  cells  of  the  upper  layer  are  elon- 
gated into  a  membraneous  border.  Get  the  air  all  out  of 
the  breathing  places,  if  possible,  since  it  very  materially 
injures»the  image.  Branched  cell  fibres,  two  or  three  cells 
high  project  into  the  air-chamber,  arising  from  the  next 
lower  layer  of  cells  which  are  flat  and  mostly  free  from 
chlorophyll.  On  the  lower  side  of  the  thallus  at  the  mid- 
dle nerve,  are  the  lateral,  alternating,  overlapping  scales. 
Between  the  scales  lie  the  transections  of  the  bundles  of 
rhizoids.  A  middle  longitudinal  section  shows  the  inser- 
tion of  the  stronger  common  rhizoids  which  uniformly  de- 
scend from  the  thallus,  and  the  cone-bearing  rhizoids. 

Metzgeria  fwxata  (5)  is  a  very  simply  constructed  thal- 
lus and  in  many  respects  very  instructive.  The  incon- 
spicuous plant  is  widely  distributed,  and  on  the  bark  of 
deciduous  trees  is  not  diflicult  to  find.  The  thallus  is 
ribbon-shaped,  clear  green  dichotomously  divided,  and  has 
a  mid-rib  distinguishable  by  the  naked  eye.  Aside  from 
this  mid-rib  the  thallus  consists  of  one  layer  of  cells,  which 
are  polyhedric,  and  filled  with  long  chlorophyll  grains. 
The  slender  mid-rib  projects  much  more  on  the  under  than 
on  the  upper  side.  It  consists,  as  one  may  see,  by  focussing 
down  through  it,  first  of  bioad  and  but  little  elongated 
cells,  then  of  slender  elongated,  and  finally  again  of  broad 
cells.  The  two  outer  layers  contain  chlorophyll.  At  the 
vegetative  point  on  the  under  side  of  the  nerve  are  a  few 
short  club-shaped  hairs,  filled  with  a  strongly  refractive 
contents.  Out  of  the  older  parts  of  the  nerve  and  also 
out  of  the  marginal  cells  of  the  thallus  come  the  so-called 
bristle  hairs  which,  under  favorable  ciroumstauces,  form 
a  lobed  holding-disk  and  thus  serve  as  rhizoids.  They 
are  always  placed  on  the  posterior  end  of  the  cell  from 


APICAL   SINUS    OF   LIVERWORTS. 


189 


which  they  are  separated  by  a  bent  division  wall  which 
does  not  pass  through  the  whole  height  of  the  cell,  but 
rather  cuts  oft*  but  a  corner  or  edge  of  it.  The  inner  cells 
of  the  mid-rib  are  somewhat  strongly  thickened,  with  al- 
most colleuchyma  sparkling- white  walls.  The  dividing 
process  at  the  vegetative  point  may  be  followed  in  Metz- 
geria  in  the  easiest  and  most  instructive  manner  (6).    In 


Fig.  71.  Terminal  growth  of  Metzgeria  furcata.  t,  apical  cell;  s'—s'",  series  of 
consecutive  segments;  m'  and  m",  marginal  cells  of  the  first  and  second  order; 
p\  flat  or  outer  midrib  cells  of  the  first  order;  ii,  inner  cells  of  the  midrib;  c,  club- 
shaped  hairs.  The  picture  is  made  with  the  lens  focussed  on  the  inner  cells  of  the 
midrib.  X  510. 

the  Metzgeria  the  growing  point  is  but  very  slightly  re- 
entrant. The  bottom  of  this  apical  sinus,  exactly  at  the 
point  where  the  mid-rib  ends,  will  be  occupied  with  the 
apical  cell.  Examine  it  from  above  so  as  not  to  be  dis- 
turbed by  the  club-shaped  hairs.  The  apical  cell  is  two 
edged.  Fig.  11,  t,  and  has  the  form  of  an  isosceles  triangle, 


190  APICAL   SINUS    OF   LIVERWORTS. 

with  the  base  towards  the  front,  mostly  somewhat  convex 
and  the  sides  slightly  bent.  It  is  divided  by  walls  which 
run  parallel  to  the  lateral  walls  and  gives  off  segments  al- 
ternately right  and  left,  which  consequently  all  lie  in  one 
plane. 

Notes. 

(1)  See  P.  G.  Lorentz,  Jahrb.  f.  wlss.  Bot.,  Bd.  vi,  1867-8,  p.  3G3; 
Goebel,  Grundriss  der  systematischen  und  speciellen  Pflanzenmor- 
phologie, 1882,  p.  184;  there  also  the  literature  p.  179.  Later  studies 
also  G.  Fritsche,  Ber.d.  deutsch,  bot.  Gesell.,  i  Jahrg.,  p.  83  und  Hab- 
erlandt;  the  same,  p.  263. 

(2)  See  Leitgeb,  Untersuchung  über  die  Lebermoose,  \i  Heft, 
1881.     There  the  rest  of  the  literature. 

(3)  Pfeffer,  die  Oelkörper  der  Lebermoose,  Flora  1874,  No.  2. 

(4)  Voigt,  Beitrag  zur  vergl.  Anat.  der  Marchantien,  Bot.  Zeitg. 
1879,  sp,  729. 

(5)  See  Leitgeb,  quoted  above,  Heft  in,  p.  34.  There  also  the 
other  literature. 

(6)  See  Kny,  Jahrb.  f.  wiss.  Bot.  Bd.  iv,  p.  85. 


LESSON  XIX. 

Histology  of  the  Fungi,  Lichens  and  Alg-^. 
Staining  the  Cell  Contents. 

The  vegetative  organs  of  the  Fungi,  with  the  exception 
of  a  few  of  the  simplest  forms,  consist  of  threadlike,  elon- 
gated, more  or  less  elaborately  branched  cells,  the  "hyphfs  " 
so  called.  They  are  either  with  or  without  division  walls. 
The  most  massive  funo-us  bodies  consist  of  aijijre orations  of 
this  hyphse.  Ttie  hyphse  may  really  be  so  solidly  united 
into  a  mass  as  to  form  a  tissue,  called  pseudoparenchyma 
which  very  strikingly  imitates  the  appearance  of  the  par- 
enchymatous tissue  of  the  higher  plants.  Still  this  pseu- 
doparenchyma is  the  product  of  a  union  of  cell  fibres  and 
not  of  the  progressive  division  of  cells  in  three  directions. 
For  the  study  of  this  kind  of  structure  we  will  take  the 
fruit  body  of  a  toadstool,  Agaricus  campestris  (1) ,  a  plant 
found  the  year  round  and  of  comparatively  simple  struct- 
ure. First  make  a  longitudinal  section  of  the  pedicel  of 
a  full  grown  plant.  We  find  a  structure  of  longitudinally 
running  hyphse,  which  we  can  easily  unravel  in  that  direc- 
tion with  a  needle.  The  hyphae  are  arranged  more  or  less 
parallel  with  each  other,  single  ones  occasionally  run- 
ning obliquely  between  the  others.  Each  hypha  forms  a 
cell  thread  which  is  laterally  branched,  here  and  there  by 
branches  which  spring  from  under  the  division  walls  or 
else  farther  down  alonof  the  side.  Sometimes  the  cells  of 
neighboring  hyphse  are  connected  by  a  cross  branch,  and 
communicate  openly  with  each  other.  At  the  periphery 
of  the  stem  the  hyphee  are  slenderer,  more  closely  com- 
pacted together  and  on  the  surface  their  walls  are  brown, 
and  their  inner  cavity  more  or  less  perfectly  collapsed. 

(191) 


192  STRUCTURE    OF   FUNGI. 

Towards  the  middle  of  the  pedicel  the  hyphas  become  like- 
wise slenderer  but  their  texture  is  more  loose  and  their 
course  altogether  irregular.  Large  quantities  of  air  fill 
the  spaces  between  the  hyphae.  Until  the  disturbing  in- 
fluence of  the  water  on  the  cell  contents  has  made  itself 
felt,  little  is  to  be  remarked  of  it ;  sometimes  on  the  trans- 
verse walls  a  collection  of  it  may  be  seen.  Afterwards 
large  vacuoles  form  in  the  cells.  Infrequently  small  crys- 
tals may  be  found  in  the  cells. 

A  transection  of  the  pedicel  gives  a  parenchymatous  ap- 
pearance which  is  lost  only  in  the  middle 
of  the  section  where  the  hyphse  present 
their  sides  to  view.  This  pseudoparen- 
chymatous  tissue  appears  to  be  formed 
from  cells  of  various  sizes  irregularly  po- 
lygonal, with  more  or  less  numerous  in- 
FiG.  72.  Agaricus^^^'^^^^^^^^'  spaccs  and  Openings  between 
cavipestris.  Part  of  a  them.     Fig.  72.     Sometimcs  the  section 

transection  of  the  ped-  ,  n      . 

icei.  Two  of  the  hy-  Will  cut  closc  to  the  trausvcrsc  wall    in 

ph.-e  are  cut  near  the  ^j^-^j^  ^^gg  ^         jj^j.  ^^-^^  ^^  g^^^  j^^  ^j^g  ^^^^ 
division   wall,   a    dot  '■ 

appearing  in  tiiem.  X  die  of  the  cell.  Scc  Fig.  72.  It  is  a  pit 
which  is  covered  on  each  side  of  the  cell 
wall  with  a  small  collection  of  light-refracting  substances. 
Such  pits  in  the  centre  of  the  cell  wall  are  quite  common  in 
the  Basidiomycetece  and  the  Ascomyceteoe  (2).  In  the  pro- 
toplasmic wall-lining  of  the  cells  are  numerous  very  small 
nuclei  but  Avhich,  not  being  easily  seen,  we  will  not  fur- 
ther consider. 

For  a  knowledge  of  the  structure  of  the  frond  of  the 
lichens  Ave  will  select  Physcia  ciliaris  found  on  tree- 
trunks  everywhere.  The  thallus  is  an  ascendant  leafy 
bush,  on  the  back  gray  green  to  living  green,  on  the  front 
gray.  Stiff  hairs  grow  from  the  edges,  are  often  forked 
and  when  they  reach  the  substratum  grow  to  it.     Make  a 


STRUCTURK  OF  LICHENS.  193 

section  between  pieces  of  elder  pith  and  examine  with  a 
sufficiently  high  power,  and  we  shall  see  that  the  thallus 
consists,  on  the  back  side,  of  a  compact  layer  of  narrow, 
thick-walled  hyph;e,  the  rind  layer.  Farther  inward  the 
hyplue  wind  about  each  other  in  order  to  make  the  loose 
tissue  of  the  fundamental  layer.  Here  it  is  easy  to  de- 
monstrate that  the  hyphjs  are  long,  branched  tubes  jointed 
by  division  walls.  On  the  border,  between  the  rind  and 
pith,  are  comparatively  large  green  round  cells,  the  goni- 
dia.  They  are  the  same  as  the  algas  Cystococcus  humicola 
Nägl. 

The  hyphffi  lie  about  the  gonidia  and  carry  to  them  raw 
nutritive  substances,  of  which  they  receive  back  a  portion 
when  it  has  been  assimilated  by  the  gonidia.  There  is 
here,  therefore,  a  case  of  communal  life  between  the  fun- 
gus and  the  alga  by  which  they  are  mutually  serviceable  to 
each  other.  On  the  underside  of  the  thalkis  the  hypha3 
are  in  this  species  again  closely  interlaced  to  form  a  lower 
rind,  or  the  loose  fundamental  tissue  extends  to  the  lower 
surface,  the  latter  being  the  most  prevalent  case  ;  but,  on 
the  edges,  the  rind-layer  of  the  back  of  the  frond  passes 
around  to  the  front  side,  in  all  cases.  From  these  edges 
the  hold-fasts  or  rhizines  grow,  consisting  of  parallel  h}'- 
phse  closely  fastened  together.  The  walls  of  these  hyphte 
are  brown.  This  string  of  til)res  is  often  divided  at  the 
base.  In  other  lichens,  these  rhizines  grow  from  the  low- 
er surface  of  the  thallus.  Chloriodide  of  zinc  solution  im- 
mediately colors  the  gonidia  a  beautiful  blue,  while  the 
hyph^  take  only  a  yellow  or  a  yellow-brownish  color,  the 
so-called  fungfi-cellulose  reaction. 

The  thallus  of  the  plant  before  us  is  said  to  be  hetero- 
o:eneous  because  the  »onidia  are  distributed  in  a  distinct 
layer.  The  more  highly  organized  lichens  have  a  homo- 
geneous thallus,  the  gonidia  being  evenly  dispersed  through 

13 


194 


STRUCTURE  OF  FRESH- WATER  ALG^. 


the  whole  frond.  Among  the  latter  are  the  gehithious 
lichens,  in  which  the  gonidiaare  embedded  in  a  gelatinous 
jiiass  through  which  the  hyphse  of  the  fungus  freely  pene- 
trate.     The  algre  which  participate  in  the  formation  of 

the  lichens  are  of  different  species, 
are  green  or  blue  2:reen,  but  be- 
long  exclusiyeiy  to  the  lowest 
groups  of  these  plants. 

The  öladophora  (3)  furnish  us 
a  much  branched  green  thread 
whose  thickness  diminishes  with 
the  degree  of  its  branching.  It 
is  a  fresh-water  plant  widely  dis- 
tributed and  every  species  is 
adapted  to  the  investigation.  The 
determination  of  species  in  this 
genus  is  very  difficult.  We  will 
select  the  dark  green,  floating, 
tuft-forming  CladopJiora  glomer- 
ata,  for  particular  study.  It  is 
branched  in  a  bushy  form,  the 
lateral  branches  springing  from 
the  upper  end  of  the  cell.  The 
branching  is  acropetal  so  that  the 
end  cell  of  the  branch  may  be 
considered  as  the  apical  cell.    But 

Fig.  73.    Cladophora  glomerata.  •  i  /• 

A  cell  from  a  iiiament  prepared  there  arisc  also  troui  the  oldcr 
,with  chromic  acid  and  carmine.  '  '^^^  additional  brauchcs,    iu  a 

11,  nucleus;   cvt,  chromatoplioies;  '' 

2),  amyium-ceiiues;  a,  starcii-  certain  scusc  adveutivc  branches. 
^''^'"^'        ■  By  sufficiently  powerful  magnifi- 

cation the  wall  lining  is  seen  to  be  formed  of  small  phites. 
Fig.  73,  ch,  which  are  laterally  separated  by  delicate  col- 
orless lines.  In  each  plate  are  several  colorless  granules, 
a,  besides  which  in  several  of  the  plates  are  relatively 


STRUCTURE    OF    FRESH-WATER    ALGiE.  195 

larger,  globular,  strongly  refractive  forms,  in  which  are 
to  be  distingiiishecl  an  inner  nucleus  and  an  outer  enve- 
lope, formerly  called  amylum  centres,  but  more  recently 
"pyrenoids,"j9.  (4)  The  cells  are  filled  with  cell-sap  and 
divided  into  irregular  polygonal  chambers  of  various  sizfes, 
by  colorless  extremely  thin  plasma  plates  which  extend 
from  the  wall  layer  through  the  cell  cavity.  These  plates 
sometimes  contain  chromatophores.  By  careful  focussing, 
colorless  plasma  balls  may  be  seen  on  the  wall-layer  pro- 
jecting into  the  cell-cavity.  They  are  nuclei  in  which, 
with  a  specially  favorable  position,  nucleoli  may  be  made 
out.  In  this  plant  we  have  a  multinuclear  cell.  If  now 
we  press  upon  the  preparation  pretty  smartly,  we  shall 
see  the  wall  laj'er  forced  back  a  little,  and  the  chlorophyll 
plates  separated  from  each  other  and  rounded  out.  At  the 
same  time  the  small  grains  and  amylum  centres  stand  out 
distinctly  in  the  chromatophores,  which  now  seem  to  be 
aflected  b}'  the  water  the  same  as  the  chlorophyll  grains 
of  the  higher  plants.  If  we  now  add  a  solution  of  potas- 
sium iodide  of  iodine  the  small  grains  and  also  the  outer 
covering  of  the  amylum  centres  will  be  tinged  violet,  l)ut 
in  the  green  chromatophores,  and  also  the  occasionally 
visible  nuclei,  a  brown  color  appears.  We  must  not  fail 
to  seek  in  this  preparation  for  uninjured  cells,  in  which 
starch  grains  and  amylum  centres  are  stained  and  stand  out 
sharply  in  their  natural  position  and  in  which  also  by 
deeper  focussing  we  distinctly  make  out  the  nucleus.  Suf- 
ficiently strong  magnification  gives  us  the  angular  forms  of 
the  albumen  crystals  (5),  of  which  two  will  sometimes  be 
found  in  an  amylum  centre.  In  a  short  time,  in  the  chlo- 
rophyll plates,  are  seen  irregularly  formed  brown  grains, 
which  come  from  the  disintegrated  chlorophyll  coloring 
matter,  and  present  us  the  hypochlorine,  or  clilorophyll 
reaction  (6).     The  reaction  may  be  had  from  the  influence 


196  FIXING    AND    STAINING    ALG^. 

of  other  acid  salts, — but  we  must  adopt  other  processes 
for  studying  the  nucleus  more  exactly,  and  getting  a  look 
at  its  method  of  parting.  This  will  give  us  the  best  op- 
portunity to  get  acquainted  with  some  approved  methods 
of  fixino;  and  staininsr,  to  which  histological  studies  owe 
so  much  in  recent  times.  Put  parts  of  the  plant  in  1% 
solution  of  chromic  acid,  in  concentrated  picric  acid,  in  1% 
solution  of  chromic  and  acetic  acid,  0.  7^  of  the  former 
and  0.  3%  of  the  latter,  respectively  (7).  Let  the  tirst 
and  last  stand  several  hours.  No  harm  will  come  in 
twenty-four  hours.  The  second  may  stand  twenty-four 
hours.  Then  wash  carefully  in  distilled  water.  They  may 
be  kept  in  water  for  a  whole  day  changing  frequently.  The 
picric  acid  preparation  requires  very  careful  handling  if  it 
is  to  be  stained  with  hainatein  ammonia.  The  variously 
fixed  and  well  washed  preparation  we  now  lay  in  Beale's 
carmine  in  watch-glasses  (8),  in  Thiersch's  or  Grenacher's 
borax-carmine,  and  in  Hoyer's  neutral"  carmine.  The 
plant  should  be  subjected  to  the  action  of  Beale's  carmine 
twenty-four  hours,  half  that  time  to  Hoyer's,  several  hours 
in  the  borax-carmine.  Another  part  of  the  plant  we  will 
stain  with  Grenacher's  or  Boehmer's  haematoxylin  which 
to  stain  well  should  be  as  old  as  possible  and  should  be 
used  very  dilute.  It  is  best  to  test  the  staining  from  time 
to  time  by  examining  with  the  microscope,  and  when  the 
requisite  degree  of  intensity  is  reached  to  take  it  out  of 
the  solution.  If,  in  spite  of  this  precaution,  the  color  be- 
comes too  dark,  it  may  be  put  in  pure  water,  or  in  a  solu- 
tion of  alum,  or  in  water  containing  a  trace  of  muriatic 
acid,  till  the  required  shade  of  color  is  obtained.  If  acid 
is  used  in  removing  the  color,  the  specimen  should  be 
afterwards  transferred  to  a  weak  solution  of  ammonia  for 
a  few  minutes.  In  order  to  stain  the  preparation  with  the 
ammoniacal  hamatem  method  (9)  we  must  remove  the  last 


FIXING   AND    STAINING   ALG^.  197 

trace  of  the  picric  acid  by  putting  it  in  a  large  quantity  of 
well  boiled  water,  which  we  repeatedly  change,  for  from 
twenty-four  to  forty-eight  hours.  For  the  preparatiou  of 
the  staining  fluid  we  throw  some  ha^matoxylin  crystals  into 
a  small  quantity  of  distilled  water  and  blow  upon  it  a  jet  of 
ammonium  gas.  This  is  done  by  means  of  a  wash  bottle 
containing  an  ammonia  solution,  in  which  the  two  glass 
tubes  do  not  reach  the  fluid.  The  crystals  dissolve  with  a 
beautiful  violet  color.  Dilute  the  solution  with  distilled 
water  and  let  it  stand  two  hours.  The  right  shade  of  col- 
or may  be  determined  directly,  but  it  is  well  perhaps  to 
make  the  color  too  high  and  weaken  it  bj'  inmiersion  in 
water  for  several  hours.  This  method  of  staining  requires 
care  but  it  gives  the  most  satisfactory^  results.  Prei^arations 
hardened  with  anything  else  than  picric  acid  are  less  suited 
to  this  staining.  The  other  named  carmine  stains  are  most 
beautiful  if  they  are  over  colored  and  then  laid  for  some 
time  in  a  watch  glass  with  50  to  70%  alcohol,  to  which  a 
drop  of  hj'drochloric  acid  is  added.  For  this  purpose  one 
may  keep  on  hand  a  ^%  solution  of  hydrochloric  acid  in 
70%  alcohol.  If  the  preparation  has  a  more  or  less  diffused 
stain,  the  addition  of  the  acidulated  alcohol  will  give  it  a 
sharp  stain.  The  preparatiou  should  alwaj's  be  washed 
in  alcohol  after  treatment  with  the  acid-alcohol. 

If  we  wish  to  make  permanent  preparations  of  our  stained 
objects  we  will  select  for  the  carmine  preparations  glycer- 
ine or  glycerine-jelly  or  Hoyer's  mounting  fluid.  If  we 
use  the  glycerine  or  the  glycerine-jelly  for  haematoxylin 
stains  we  must  be  sure  it  contains  no  trace  of  acid.  The 
Hoyer  mounting  fluid  is  also  well  suited  to  hsematoxyliu 
stains.  The  preparation  should  not  be  put  directly  into 
the  mounting  fluid,  else  the  cells  will  collapse  by  the  too 
sudden  withdrawing  of  the  water,  but  it  should  flrst  i)e  put 
in  very  dilute  glycerine  which  concentrates  slowly  by  stand- 


198  FIXING   AND    STAINING   ALG^. 

ing  in  the  air  where  the  water  may  evaporate.  Then  the 
plant  may  be  transferred  to  glycerine  or  glycerine-jelly  or 
Hoj^er's  mounting  fluid  without  damage.  The  glycerine 
preparation  should  he  cemented  with  Canada  balsam.  The 
other  media  named  will  need  no  cementing. 

Considerino^  now  the  different  fixino;  and  stainin«'  media 
for  pre[)arations  we  may  in  general  say,  that  chromic  acid 
or  its  mixture  goes  best  with  the  carmine  stain  ;  and  picric 
acid  for  fixing  with  the  haematoxylin  or  the  hamatein  am- 
monia staining  fluid.  But  it  must  be  expressly  emphasized 
that  these  results  are  restricted  to  the  present  ol)ject,  and 
that  other  objects  may  be  better  treated  by  other  methods. 
It  also  frequently  happens  that  an  already  tested  staining 
fluid,  for  some  unknown  reason  fails,  so  it  will  not  be  safe 
to  base  a  conclusion  on  a  single  case.  Generally  the  fix- 
ino-  and  stainins:  <>f  cell  contents  is  an  art  which  can  be 
learned  onl}'  by  practice,  and  one's  first  attempts  are  often 
failures.  We  have  chosen  the  Cladophora  as  one  of  the 
most  suitable  objects  for  the  introduction  of  the  different 
hardening  and  staining  processes.  He  who  will  f()lh)w  the 
method  given  here,  strictly,  will  seldom  fail  ;  hardening 
with  ]%  chromic  acid,  and  staining  part  with  borax-car- 
mine, and  anothar  with  hsematoxylin. 

In  the  borax-carmine  preparation.  Fig.  73,  the  nucleus 
comes  out  very  sharply.  The  amylum  centres  and  the  rest 
of  the  cell  plasma  remain  as  good  as  uncolored  and  the 
starch  grains  take  no  color.  AVithin  the  amyhun  centres, 
the  albuminous  crystals  are  quite  distinct  surrounded  by 
a  hollow  globe  which,  as  we  saw,  gave  the  starch  reaction 
with  iodine.  The  nuclei  are  distributed  quite  uniformly 
through  the  cells  lying  in  the  chlorophyll  layer  and  project- 
ing into  the  cell-mass.  The  nucleus  shows  a  darkly  stained 
nucleous  and,  for  the  rest,  seem  to  be  finely  granular  or 
minutely  porous.     The  hsematoxylin  or  hamatein  prepara- 


CULTIVATING    FR:^SH-WATER    ALG^. 


199 


tions  have  the  nucleus  colored  dark,  also  the  crystals  in 
the  amjlum  centres,  even  if  but  slightly.  The  starch  grains 
are  not  colored,  but  the  microsomes  of  the  cell-plasma  are, 
and  almost  as  the  crystals. 

The  genus  Spirogyra  presents  us  with  a  simple  filamen- 
tous cell.  We  will  choose  a  species  which  has  a  central, 
ea!<ily  visible  nucleus,  Spirogyra  majuscula,  which  is  spo- 
radic but  not  rare  in  ponds.  Other  species  with  central 
nucleus  will  serve  and  their  essential  structure  is  about  the 
same.  We  should  make  a  culture  of  our  material.  Use  a  rel- 
atively low  vessel  whose  walls  are  untransparent  or  may  be 
made  so  by» putting  black  pa- 
per around  them,  as  lateral  il- 
lumination is  damairino;  to  the 
plant.  The  vessel  should  be  set 
in  a  bright  place  but  be  pro- 
tected from  direct  sunlight. 
Into  the  river  or  spring  water 
throw  from  time  to  time  bits 
of  turf  boiled,  and  saturated 

.  ,  .  ^.       .,  Fig.  74.    Spirogyra  majuscula,  a  cell  o( 

Wltn  a  nutrient  liquid  com-  ^  f5i_^„,e„t;  gj,o^r,  l^y  (different  rocnsslng, 
pounded   as  follows  :    To     100   ^^l*"  representing    the    central  nucleus 

and  its  suspending  threads.  X  "-^lO- 

cc.  of  water  add  1  g.  nitrate 

of  potash,  0.5  g.  common  salt,  0.5  g.  sulphate  of  lime,  0.5 
g.  sulphate  of  magnesia,  a  trace  of  finely  pulverized  phos- 
phate of  lime  (11).  Spyrogyra  and  other  fresh-Avater  algse 
will  flourish  well  under  such  conditions.  The  cells  of  the 
filament  are,  when  full-grown,  about  1^  to  2  times  longer 
than  broad,  Fig.  74.  The  cell  membrane  is  lined  with  a  del- 
icate colorless  protoplasmic  layer  which  becomes  clearly 
visil)le  when  the  protoplasmic  cell  body  is  made  to  contract 
by  withdrawing  the  water  from  the  cell  by  means  of  glyc- 
erine, a  solution  of  sugar,  cooking  salt,  or  saltpeter.  To 
the  protoplasmic  layer  succeed  eight  to  ten  chlorophyll 


200  STRUCTURE    OF    FRESH- WATER    ALGiE. 

bands  which  appear  to  be  wound  steeply  and  closely  abont 
the  cell.  The  bands  are  considerably  indented,  and  are 
sufficiently  transparent  to  allow  us  to  look  into  the  interior 
of  the  cell.  Thick,  ronnd  colorless  bodies,  anndum  cen- 
tres, are  embedded  in  the  bands  at  irregular  intervals.  In 
these  are  the  albuminous  crystals  and  a  surrounding;  layer 
of  small  starch  grains.  The  crystals  may  be  seen  without 
reagents.  They  come  out  most  distinctly  when  alcohol 
acidulated  with  picric  acid  is  added  at  the  edge  of  the  cov- 
er-glass. Treatment  with  potassic  iodide  affects  alike  the 
color  of  the  crystals  and  the  starch  membrane  making  the 
whole  body  a  dark  brown.  The  central  nucleus  is  spindle- 
shaped  in  this  si)ecies.  JNIoved  out  of  position  by  pressure 
it  is  seen  to  be  disk-shaped  from  the  front,  so  it  in  reality 
has  the  form  of  a  biconvex  lens.  In  its  centre  lies  a  largo 
distinct  nucleolus  ;  sometimes  two  or  even  three  of  these 
mny  be  seen  in  the  nucleus.  In  other  nearly  related  spe- 
cies the  nucleus  is  thicker  and  in  its  natnral  position  in  the 
cell  seems  right-angled  with  the  angles  rounded  off.  Tiie 
nucleus  is  surrounded  by  a  very  thin  plasma  layer  from 
which  delicate  threads  of  protoplasm  run  out  to  the  wall 
layer  of  the  cell.  Tlie  nucleus  is  suspended  by  these 
threads  in  the  cell-sap  which  fills  the  cell.  The  threads 
spring  from  all  of  the  sharp  edges  of  the  nucleus,  mostly 
repeatedly  fork  in  their  course  and  fasten  themselves  to  the 
inside  of  thechlorophyll  bands  particularly  to  the  projecting 
places  which  cover  the  amylum  centres.  ■  This  may  all  be 
seen,  in  most  cases,  by  slowly  changing  the  focus. 

Notes. 

(1)  H.  Hoflman,  Icoiies  anal,  fniig.,  i— iii;  de  Baiy,  Morph,  d.  Pilze 
etc.   p.  49  ff. 

(2)  Ueber  die  Tapfel  in  den  Scheidewänden  der  Florideen,  vergl. 
Bornet,  Etudes  phycol.,  p.  100  und  Schmitz,  Stzber.  d.  kgl.  Akad.  d. 
Wiss.  z.  Berlin,  1883,  p.  218. 


LITERATURE    OF    THE    PRESENT    LESSON.  201 

(3)  Schmitz,  Siphoiiocladiaceen,  p.  17;  Strasburger,  Zellh.  unci 
Zellth.,  Ill  Aufl.,  p.  204. 

(4)  Schmitz,  Chromatophoren  d.  Algen,  p.  37.     See  also,  pp.  16  u.  35. 

(5)  Nach  Mittheilungen  von  A.  W.  Schimper. 

((i)  Pringsheim,  besonders  in  den  Jahrb.  f.  wiss.  Bot.  Bil.  xii,  p. 
294;  A.  Tschirsch,  Ber.  d.  deut.  bot.  Gesell.  Bd.  i,  p.  140.  There  the 
literature. 

(7)  Fleming,  zuletzt  in  Zcllsubstanz,  Kern  und  Zelltheilung,  1882,  p. 
379.     There  also  the  literature. 

(8)  The  capacity  of  the  nucleus  to  absorb  coloring  matter  with  avid- 
ity was  discovered  by  Thomas  Hartig  and  published  in  Bot.  Zeitg  , 
1854,  Sp.  877,  under  the  title  of  "  Ueber  das  Verfahren  bei  Behandlung 
des  Zellkorns  mit  Farbstoffen."  Entwickelungsgeschichte,  d.  Pflanz- 
keims, 1858,  p.  154.  In  animal  histology  the  experimt-nts  of  Gerlach 
should  be  quoted.  Mikr.  Stud.  a.  d.  Geb.  d.  menschl.  Morpholg..  1858. 

(9)  See  Schmitz,  Stzber.  d.  niederrh.  Gesellsch.,  13  Juli,  1880,  Sep. 
Abdr.,  p.  2. 

(10)  Strasburger,  Zellb.  u.  Zellth.,  m  Aufl.,  p.  173. 

(11)  Niüirstofflösuugi  nach  Sachs  Vorl.  über  Pflanzeu-Physiol.,  p.  342. 


LESSON  XX. 

Diatoms,  Protococcus,  Yeast,  Photophytes. 

The  diatoms  or  bacillaria  are  single-celled  organisms 
which  form  a  definite  group  by  themselves,  and  occupy  an 
intermediate  position  between  animals  and  plants.*  Pin- 
nularia  viridis,  a  species  often  occurring  in  standing  or 
running  water,  gives  us  a  most  suitable  object  by  which 
to  examine  the  structure  of  the  diatoms  (1).  The  fresh- 
water forms  attain  considerable  size  and  so  generally  give 
us  a  ready  view  of  the  structural  relations  of  their  organ- 
isms. With  our  highest  available  magnification,  they  np- 
pear  either  as  an  elongated  ellipse  or  as  a  rectangular  ol)ject 
Avith  somewhat  rounded  corners.  In  the  former  case,  we 
get  a  side  view,  Fig.  75  A,  and  in  the  latter  a  front  vieAV, 
Fisf.  75B.f  In  the  side  view  we  see  the  cell  membrane 
marked  by  slender  grooves  which  run  from  the  edge 
toward  the  middle  but  not  quite  to  it.  They  are  mostly 
held  to  he  flutings  or  depressions  in  the  surface  of  the 
shell,  thin  places  in  the  sul)stance  of  the  frustule.  The 
middle,  smooth  surface  left  by  the  grooves  has  at  its  ends 
and  also  in  the  middle,  strongly  refractive  thickenings, 
desio-nated  nodules.  The  two  terminal  nodules  are  c(m- 
nected  with  the  middle  one  by  a  line  which  close  to  the 
central  nodule  bends  aside  l)()th  the  same  way  and  ends 

*This  statement  will  greatly  sui-pvise  my  readers,  I  imagine,  coming  from  so 
eminent  a  botanist  as  Dr.  Straslnirger.  Tliat  diatoms  are  plants,  and  plants  too 
not  of  tlie  lowest  class,  there  is,  I  suppose,  no  good  reason  to  doubt.  Saclis  places 
them  among  the  Zygofporece  after  the  desniids.  See  Lehrbucli  d.  Botanik,  viei'te 
Auflage,  p.  '2G4,  English  Edition,  p.  21)0.  Tliwaites  tiist  discovered  tlie  se.\ual  re- 
production of  diatoms,  by  conjugation,  lorty  years  ago.  See  Ann.  Nat.  Hist., 
1847— A.  B.  ir. 

t  In  general,  diatoms  are  said  to  present  a  "front"  view,  when  the  side  having  the 
suture  is  turned  towards  us.  The  side  view  is  when  we  look  upon  llie  broad  side 
of  the  frustule.— A.  B.  H. 

(202) 


STKUCTUEE    OF    DIATOMS. 


203 


with  a  slight  thickening.  The  tenninal  nodules  are  in- 
closed by  the  ends  of  these  lines,  which  make  a  crescent 
about  one  side  of  them  in  the  same  direction  as  about  the 
middle  nodide.  BetAveen  the  nodules  the  lines  widen  a 
little  as  if  they  had  an  opening  towards  the  inside  of  the 
cell.  In  a  front  view  the  grooves  are  seen  onl}^  on  the 
edges  of  the  frustule.  See  £,  Fig. 
75.  By  focussing  on  the  optical 
diameter  of  the  cell  and  carefully 
observing  the  ends,  we  see  that  a 
middle  strip  of  the  wall  is  double. 
By  a  thorough  examination  we  find 
that  one  part  of  the  wall  here  shuts 
over  the  other.  On  the  sides  of  the 
two  elliptical  parts  of  the  wall  which 
we  saw  in  the  side  view,  is  fixed  a 
membranous  portion  which  ends  in 
a  free  edge.  The  walls,  then,  of 
this  cell,  consist  of  two  halves,  one 
of  which  is  inclosed  in  the  other,  and 
the  whole  cell  resembles  an  elliptical 
box  with  a  cover  shuttinof  down  over 
the  top  of  it.  If  we  now  pass  from 
the  optical  diameter  of  our  cell  to  the 

„.,,,,,  j4    :  ,1   ..:    .  ,     , r   ii  j-i  Fig.  75.    Pinimlnria  viridis. 

superhciai  view,  we  can  to    ow  the    ,     .        ,.  ^,       .,      ,.  ,, 

i  '  A,   view  Ol    the    side  ot    the 

line  edges  of  the  two  halves  of  the  ivustuie.  tj,  view  of  the  front 

1,      ,  1    !•       ^       !•  T        1  •      with  the  central  surrounding 

cell,  here,  as  delicate  lines.    In  this  baud.  xsio. 

genus  it  is  easy  to  separate  the  two 

parts  of  the  frustule  by  pressure  or  by  chemical  reagents. 

Examples  may  also  be  found  in  which  the  dead  plants  have 

more  or  less  fully  undergone  this  process.     Pressure  also 

will  crack  the  frustule  along  a  line  parallel  Avith  the  edge  of 

the  overlapping  part,  but  a  little  distance  from  it.     There 


204  STRUCTURE    OF   DIATOMS. 

may,  therefore,  be  two  of  these  Ihies  which  may  be  sup- 
posed to  be  thin  places  in  the  frustule.  They  do  not  ex- 
tend to  the  ends  of  the  cell.  The  contents  of  the  cell  also 
have  a  different  appearance  in  the  two  views.  In  the  first, 
Fig.  75  A,  a  clear  stripe  extends  through  the  middle  of  the 
cell  from  one  end  to  the  other.  The  colorless  cytoplasm 
of  the  cell  is  consequently  visible.  Near  the  middle  of  the 
cell  is  a  biconcave  plasma-bridge.  In  this  bridge  is  the  nu- 
cleus provided  with  large  nucleoli,  not  always  visible  with- 
out the  help  of  reagents.  Adjacent  to  the  clear  stripe  on 
both  sides  are  the  endochrome  plates,  the  brown-colored 
chromatophores,  lying  upon  the  overlapping  parts  of  the 
frustule.  In  the  plasma-bridge  are  slender  rods,  connected 
in  pairs,  of  unknown  significance.  In  the  cell-sap  are 
usually  oil-drops  of  various  sizes.  In  the  front  view.  Fig. 
75,  B,  the  cell  body  appears  uniformly  brown  because  the 
chromatophores  cover  the  whole  colorless  wall  layer.  At 
the  extreme  ends  of  the  cell  only  may  the  colorless  cell 
plasma  be  seen.  The  chromatophore  is  of  uniform  thick- 
ness and  color  throughout.  In  this  view,  also,  the  central 
plasma  collection  takes  the  form  of  a  biconcave  bridge. 

If,  now,  we  examine  the  Cladophora  preparation,  most, 
likely  we  shall  find  some  diatoms  attached  to  that,  which 
have  been  fixed  and  stained  at  the  same  time   with  the 
alga,  and  show  the  nucleus  most  beautifully. 

We  shall  often  find  among  Plnnularia  many  double 
compound  forms.  They  are  sister  frustules  produced  by 
the  self-division  of  the  mother  plant.  The  sides  of  the 
frustules  adhere  to  each  other,  and  if  the  walls  of  the  two 
plants  are  already  quite  full  grown,  we  shall  find  that  the 
two  outer  halves  of  the  two  frustules  shut  over  the  inner 
halves.  By  the  parting  of  the  contents  of  the  mother 
cells,  these  inner  walls,  for  each  daughter  cell,  are  pro- 


STRUCTURE    OF   DIATOMS.  205 

diiccd.  Euch  cell  possesses,  therefore,  an  older  jiiid  a 
younger  wall,  and  the  difference  of  their  ages  may  be  very 
considei'able. 

The  Pinnularia  specimens  may  be  seen  in  motion.  The 
cells  proceed  commonly  in  the  direction  of  their  longer 
axis,  but  may  also  sometimes  turn  aside  from  their  path. 
They  do  not  swim  freely  through  the  water,  but  creep 
over  the  surface  of  the  substratum  ;  probably  the  line 
which  we  saw  in  the  mitldle  of  tlie  frustule  is  a  cleft  in  it, 
through  which  a  protoplasmic  membrane  protrudes  and 
becomes  the  organ  of  locomotion,  a  kind  of  Pseudopo- 
dium. The  motion  is  either  uniform  or  by  sudden  move- 
ments. 

If  we  place  our  Pinnularia  preparation  on  a  piece  of 
mica  and  heat  it  red  hot  over  a  spirit  or  gas  flame,  and 
then  examine  it  on  a  slide  dry,  and  also  under  a  cover- 
glass  with  high  powers,  we  shall  see  that  the  diatoms  are 
perfectly  skeletonized.  If  the  heat  is  applied  but  a  short 
time,  the  frustules,  from  the  burning  of  the  organic  sub- 
stance, become  brown,  but  by  longer  firing  they  are  ren- 
dered colorless.  iNIuriatic  acid  will  not  touch  them.  They 
consist  of  silex  and  show  the  finest  characteristic  of  the 
structure  of  the  cell  wall.  They  must,  therefore,  be  silici- 
fied  in  the  highest  degree.  The  grooves  in  this  preparation 
show  very  distinctly  as  dark  stripes  ;  also  other  structural 
characteristics  of  the  walls  may  be  studied.  Particularly 
beautiful  are  the  lines,  seen  in  the  side  view,  running  from 
the  end  to  the  middle  nodules  ;  they  distinctly  widen  in  the 
middle.  In  the  front  view  the  edges  of  the  two  halves  of 
the  frustule  are  very  distinct,  and  the  fine  lines  also  par- 
allel to  these  edijes,  but  not  extendino;  to  the  ends.  Beau- 
tiful  skeletons  of  the  diatoms  may  also  be  prepared  by 
treating  the  living  plants  with  a  drop  of  concentrated  sul- 
phuric acid;  then,  after  a  little,  adding  first  twenty  per 


206 


STRUCTURE    OF    PROTOCOCCUS. 


cent  and  gr.'idiially  concentrated  chromic  acid  and  finally 
removing  both  with  water  (2). 

This  remarkable  construction  of  the  cell  wall  out  of  two 
distinct  parts  is  also  common  to  other  diatoms  ;  so  is  also 
the  power  of  locomotion..  Even  those  forms  which  grow 
inclosed  in  gelatinous  tubes  have  this  power  when  once 
freed,  but  it  seems  mostly  to  be  wanting  in  the  thread-like 
forms.  On  account  of  the  extraordinary  delicacy  of  the 
structure  of  the  cell  walls  of  these  plants,  their  frustules 
are  used  as  objects  for  testing  the  higher  powers   of  the 


FtG.  T*».    Protococcn    luilis    ifcci    tieitmeut  with  potassium  iodide  of  iodine. 
Ill  D  the  cells  to  tue  leiL  siiuixiy  aiier  iiaiims.     A  ^^0. 

microscope.  Pleurosigma  anguloliim,  when  subjected  to 
the  highest  magnification,  shows  the  stri«  resolved  into 
regularly  arranged  hexagons. 

We  will  study  the  Protococcus  to  learn  the  nature  of  the 
simplest  form  of  the  monocellular  green  algte.  To  this 
group  belong  all  the  green  collections  on  the  stems  of 
trees,  damp  boards  and  walls  and  other  such  situations. 
We  will  leave  the  question  in  doubt  whether  our  Proto- 
coccus is  an  independent  species,  or  is  only  a  single  stage 
of  development  of  another  alga  (3).  The  specimen, 
Fig.  76,  which  we  took  from  an   old  trunk  of  a   tree  is 


STRUCTURE  OF  PKOTOCOCCUS.  207 

Protococcus  viridis.  Usiiiij  a  high  magnificution,  we  find 
it  to  con-^ist  of  isohited,  gh)bular  cells,  or  small  groups  of 
the  same,  Fig.  76,  A-F.  The  contents  of  the  cell  are 
bright  green,  hut  the  plasma  mass  is  not  uniformly  col- 
ored, hut  by  the  highest  magnification  we  discover  a 
number  of  chiomatophores,  which,  in  mutual  contact,  oc- 
cupy the  surface  of  the  cell  contents.  As  their  contact  is 
not  perfect,  the  colorless  plasma  comes  in  view  between. 
Near  the  middle  of  the  cell  ma}'^  be  seen  —  not  often  how- 
ever without  the  help  of  reagents — the  nucleus  with  its 
nucleoli.  The  thin  cell  walls  are  colored  violet  with 
chloriodide  of  zinc.  Most  of  the  cells  are  seen  in  the 
process  of  cell  division  by  means  of  a  partition  wall 
which  halves  the  globular  cells,  Fig.  76,  D.  Neighboring 
cells  divide  in  the  same  or  in  a  direction  at  neailj-  right  an- 
gles to  the  plane  as  the  first.  The  daughter  cells  assume  a 
globular  form  when  they  pass  out  of  the  original  connection 
and  either  adhere  for  a  considerable  time  or  become  fully 
separated,  Fig.  76,  CF.  Treating  the  cells  with  potas- 
sium iodide  of  iodine  will  bring  out  the  nucleus  very 
sharply.  The  figures  in  the  illustration  are  from  an  io- 
dine preparation.  Nucleoli  will  be  clearly  visible  in  every 
cell.  In  the  newly  formed  cells  the  nucleus  will  lie  on 
the  side  of  the  partition  wall.  Fig.  76,  D.  The  iodine 
solution  detects  small  starch  grains  in  the  chromatophores, 
but  no  amylum  centres. 

We  meet  a  very  simply  constructed  organism  in  the  col- 
orless fungus  cells,  hitherto  included  in  the  Saccliaromy- 
cetoa..  Take  a  small  quantity  of  beer  yeast  from  a  well 
fermented  mash  in  a  brewery  and  examine  it  in  water  Avith 
a  high  magnification.  Our  field  of  vision  Avill  be  filled  with 
small  cells  which  are  the  individual  plants  of  the  so-called 
beer  yeast  Saccharomycis  cerevisice  (4).  The  cells  are 
globular  or  ellipsoidal,  have  a  delicate  cell-membrane,  and 


208        STEUCTURE  OF  BEER  YEAST  PLANT. 

within  one  laro;e  or  several  smaller  vacuoles  and  some 
granules  which  strongly  refract  the  light,  Fig.  77,  7.  A 
nucleus  exists  but  is  not  easily  detected  (5).  In  order  to 
see  it  we  shall  have  to  treat  the  cells  as  in  the  case  of  the 
Gladopliora  with  picric  acid  to  harden  them  and  then  stain 
with  ammoniacal  haämatein.  It  will  then  appear  near  the 
middle  of  each  cell,  small,  round,  and  darkly  stained.  As 
we  examine  the  living  object  we  shall  find  some  of  the  cells 
putting  out  one  or  more  small  buds  from  their  sides,  which 
irraduallv  attain  the  form  and  size  of  the  mother-cell  and 
then  separate  from  it.  See  Fig.  77,  ^  and  3.  In  very 
energetic  growth  we  shall  find  sometimes  a  number  of  the 
daughter  cells  connected  together,  forming  a  branched 
chain.  In  more  gradual  development  each 
iy^   @     '^^^^  ^^^^  ^^  separated  from  the  mother-cell 

^  (^  ^  before  another  starts.  This  method  of 
^  ^       propagation  gives  them  the  name  of  aS'«c- 

FiG.  77.  saccha-    cJiaromyceicd  or  budding-fungi.     In  solu- 

romyces     cereiisice.        ,  .  -  i       i      i  • 

1,  not  budding.  2, 3,  tious  oi  sugar  it  produccs  an  alcoholic 
budding  cells.  X  fermentation.  Recently  (0)  the  specific 
independence  of  the  Saccharomyceth  has 
been  denied,  and  they  have  been  explained  to  be  gonidia 
or  spores  of  different  fungi,  which  possess  the  power  in 
the  right  nutrient  solution  to  go  on  reproducing  themselves 
endlessly. 

We  will  now  turn  our  attention  to  a  JVbstoc,  which  on 
account  of  its  symbiatic  relations  to  another  plant  will  be 
of  interest  to  us.  The  latter  plant  is  Azolla  öaroliniana, 
cultivated  in  all  botanic  gardens.  Since  the  Azolla  is  win- 
tered  in  greenhouses  we  can  obtain  the  JSfostoc  at  all  times 
The  JSFostoc  generally  is  very  much  inclined  to  live  with 
other  plants  and  we  find  it  in  very  difi'erent  ones,  but  prin- 
cipally as  an  element  in  the  fronds  of  lichens.  This  fun- 
gus Anabcena  AzoUoe  may  be  found  on  a  particular  part  of 


STRUCTURE  OF  FRESH- WATER  ALG.E. 


209 


the  plant..  The  leaf  of  the  Azolla  consists  of  two  lobes  ; 
the  upper  one  is  fleshy  and  swims  on  the  water,  the  lower 
is  membranons  and  is  immersed  beneath  the  surface.  On  the 
inside  of  the  upper  leaf  is  a  cavity  which  has  an  opening 
towards  the  interior  of  the  leaf.  This  cavity  is  filled  with 
the  alga.  From  its  walls  grow  branched  hairs  between 
which  are  the  coils  of  the  alga.  In  order  to  obtain  the 
plant  for  investigation,  tear  away  the  surface  of  the  leaf 
with  a  needle  and  lay  a  cover-glass  on  it,  press  on  the  glass 
a  little  and  yon  will  be  quite  sure  to  find 
the  strings  of  the  Anabcena  on  it.  By 
examining  the  threads  with  a  high  power 
we  find  it  to  consist  of  a  series  of  barrel- 
shaped  cells,  Avhich  are  here  and  there  in- 
terrupted by  a  larger  ellipsoidal  or  round 
cell,  the  heterocyst,  or  terminal  cell,  Fig. 
78.  The  threads  are  coiled  about  snake- 
like without  visible  jelly.  The  whole 
contents  of  the  vegetative  cells  are  verdi- 
gris-green, the  terminal  cells  olive-green. 
Small  dark  granules  are  to  be  fonnd  in 
the  contents  of  the  cells  bnt  no  nncleus. 
We  often  find  the  cells  in  the  act  of  self- 
division.  Fig.  78,  a  to  d.  Take  a  twig 
between  the  fingers  and  make  a  superfi- 
cial section  of  the  leaf  and  if  the  cavity  of  the  leaf  is  cut 
just  right  we  may  see  the  Anabcena  in  its  natural  position 
— the  jointed  hairs  with  the  alo-a  coiled  amon«:  them. 

Qnite  of  the  same  structure  is  the  thread  contained  in 
the  olive-green  masses  of  jelly  Avhich  one  finds  often  in 
large  quantities  on  the  street,  and  which  belong  to  JSfostoc 
ciniflonum  (7). 

In  the  investigation  of  those  terrestrial  forms  of  Vau- 
dtevia,  especially  those  which  collect  on  flower  pots,  one 

14 


Fig.  TS.  Anabfena 
azollce-  a  to  d.  cells  in 
successive  stages  of 
self-division  ;  /(,"  a  ter- 
minal cell  or  hetero- 
cyst.   X5W. 


210 


STRUCTURE    OF   FRESH-WATEK   ALGJE, 


■will  meet  with  the  OsciUaria,  which  belongs  to  the  self- 
dividing  plants  and  is  closely  related  to  the  Nostocs.  It 
maybe  found  in  all  standing  water,  on  muddy  soil  and  such 
like  places.  An  unpleasant  mouldy  smell  often  betrays 
its  presence.  Cultivated  in  vessels  it  will  creep  up  on 
the  walls  above  the  surface  of  the  water.  It  consists  of 
nearly  straight  or  twisted  threads,  colorless  or  colored 
blue-green,  or  verdigris  or  olive-green  to  bro\vn,  which  keep 
up  a  lively  movement  among  themselves.  The  threads 
are  free  or  inclosed  in  a  gehitinous  sheath.     They  may 


Fig.  79.  A,  OsciUaria  princeps,  B,  OsciUaria  Fralichii.  a,  end  of  fllanient;  h,  a 
piece  from  the  inner  portion  of  tlie  filament.  In  B,  b,  tiie  granules  are  collected 
on  the  partition  walls  of  the  cells.  In  A,  c,  a  dead  cell  is  seen  between  the  living 
ones. 

occupy  the  sheath  singly  or  in  considerable  numbers. 
The  .shoath  arises  from  the  outer  membranous  \uyev  of  the 
ii lament.  Where  these  membranes  have  disappeared  the 
sheath  fails.  The  threads  are  divided  into  disk-like  short 
cells  by  transverse  division  walls,  the  latter  being  seen 
easily  in  some  cases  and  with  much  difficulty  in  others. 
With  the  exception  of  these  differences  a  great  uniformity 
in  the  structure  of  these  organisms  prevails.  The  contents 
of  the  cells  are  colored  throughout.  There  is  no  nucleus, 
but  numerous  small  grains  appear,  distributed  uniformly, 


MOVEMENT   OF   FRESH- WATER   ALG.E. 


211 


or  collected  on  the  partition  walls.  It  is  a  matter  of  in- 
difference which  species  we  select,  l)ut  it  will  be  of  some 
advantage  to  take  one  with  thick  filaments,  and  visible  par- 
tition walls  like  Fig.  79. 

The  movement  of  these  plants  is  very  interesting.  "With 
the  thicker  forms,  having  bent  ends  and  distinct  grains, 
and  with  a  snfiiciently  strong  magnification,  the  appearance 
can  be  fnlly  examined.  We  see  that  the  movement  of  the 
thread  is  connected  with  a  gradual  turning  upon  its  axis. 
At  the  same  time  the  thread  exhibits  irregular  bendino:s 
which  are  the  results  of  differences  in  the  intensit}^  of  the 
growth  of  its  different  sides. 
This  bending  may  go  on  very 
slowl}'  but  may  also  give  rise  to 
a  rapid  motion  when  it  is  hin- 
dered by  some  obstacle,  which 
being  overcome  the  tension  is 
suddenly  relieved.  The  move- 
ments of  the  Oscillaria  are  for- 
wards   and    backwards.       It    can       Vig.SQ.  Glceocapsapolydermatica. 

take  place  only  when  the  threads  i°  ^  "'^  seit-divisiou  is  beginning 

.  ^  .  and  in  B  at  the  left  it  is  recently 

imd    some    point  of  resistance,   completed.  X54o. 
Tlie   motion  of    the  straight 

threads  is  the  same  as  that  of  the  bent  ones,  but  is  not  so 
easily  seen ;  for,  in  the  former  case,  a  single  granule  of 
the  cell  has  to  be  fixed  in  the  attention  in  order  to  see  the 
motion  of  the  thread  upon  its  axis.  The  cause  of  this 
motion  is  not  yet  determined  with  certainty,  but  it  has  re- 
cently been  asserted  that  it  is  produced  by  a  protoplasmic 
process  protruding  through  the  membrane  to  the  outside 
(8). 

In  the  same  class  of  organisms  as  the  JVbsiocs  and  the 
Oscillaria  are  the  still  simpler  formed  Chroococcacece  which 
we  will  stndjMn  the  widely  distributed  Gloeocapsa  species. 
Glmocapsa  pohjdermatica ,  Fig.  80,  grows  on  moist  walls 


212  LIFE   HISTORY    OF   ALG.E. 

or  rocks  and  is  distinguished  by  its  dirty-green  or  olive- 
colored  jelly  mass,  and  the  solid,  distinct  and  repeatedly- 
laminated  gelatinous  covering.  Another  species  with  less 
beautifully  laminated  jelly  inclosure  would  answer  just  as 
w^ell.  In  all  species,  within  the  gelatinous  envelope,  are  cells 
without  nucleus,  more  or  less  distinctly  granular  and  uni- 
formly colored.  By  these  characteristics  of  the  cell  bodj-, 
these  plants  are  distinguished  from  many  forms  of  the  Pro- 
tococcaceoa  which  they  outwardly  resemble,  especially  the 
Falmellacece,  for  these  have  a  nucleus  and  the  chromato- 
phores  are  separated  from  the  rest  of  the  cell  plasma.  Gloe- 
oca^sa polydermatica,  shortly  before  undergoing  division, 
is  almost  globular,  Fig.  80,  C.  It  then  begins  to  gnnv  in 
leno-th,  and  soon  to  show  a  contraction  about  the  middle, 
Fig.  80,  A,  at  which  point  a  delicate  division  wall  becomes 
visible.  The  daughter  cells  round  themselves  out  and  by 
the  swelling  of  the  division  walls,  and  the  thickening  layer 
produced  from  them,  they  become  widely  separated.  By 
the  production  of  new  gelatinous  layers  on  the  inside,  the 
older  outer  ones  become  extended  and  at  last  burst  and  are 
thrown  off  (9).  A  great  number  of  generations  are  con- 
sequently connected  in  one  common  cell  family  by  the 
gelatinous  envelope,  in  the  rupturing  of  which  the  family 
is  scattered.  One  sometimes  finds  a  single  cell  existing 
alone  by  itself,  in  which  case  it  is  usually  surrounded  by 
a  considerable  number  of  jelly-layers,  Fig.  80,  A.  In 
such  a  case  the  cell-division,  not  the  wall-thickening,  ceases. 
In  the  JSfostocs,  Oscillaria  and  Ghroococcaceaz,  the  cell- 
contents  behave  difierently  from  what  they  do  in  all  those 
plants  heretofore  examined.  While  we  have  previously 
found  the  protoplasm  differentiated  into  cell-plasma,  nu- 
cleus and  chromatophore,  we  find  here  all  these  elements 
of  the  cell  body  united  in  one  substance  (10).  Constant 
deviation  in  color  from  the  pure  green  of  the  other  plants 
requires  that  they  be  separately  classed  as  Phycochromacecß 


LITERATURE  OF  THE  LESSON.  213 

or  CyanophycecB.  The  low  grade  of  their  organization  is  in- 
dicated by  their  want  of  sexual  reproduction.  A  kind  of 
asexual  reproductive  system  is  peculiar  to  all  these  (to- 
gether also  with  other  asexual  methods),  namely  :  the  one 
by  vegetative  self-division ,  hence,  these  organisms  are  called 
dividing-algfe,  Schizophycecß  (11).  Later  investigations  in- 
dicate that  the  thread-like  ScJdzophycecß  are  in  a  position  to 
be  separated  into  globular  cells  surrounded  by  gelatinous 
envelopes,  that  is,  to  enter  upon  the  Gloeocapsa-like  condi- 
tion. A  corresponding  behavior  is  observed  already  in  the 
green  algw,  in  the  Protococcacecß  and  raises  the  question  if 
Profococcus viridishe an  independent  species.  This  ques- 
tion is  repeated  in  reference  to  the  Chroococcacece  which  is 
perhaps  only  one  stage  in  the  development  of  the  thread- 
like self-dividinof  als^x. 

Notes. 

(1)  See  Pfitzner,  in  Hanstein's  Bot.  Abh.,  Bd.  i,  Heft  ii,  p.  40  und 
Sclienk's  Handb.  d.  Bot.,  Bd.  ii,  p.  410.  In  the  first  treatise,  tlie  lit- 
erature may  also  be  found. 

(2)  Miliaralvis,  die  Verkieselung,  Wiirtzburg,  1884. 

(3)  See  on  this  point  especially  Cienkowski,  Bot.  Zeitg.,  1876,  Sp. 
17,  u.  Mel.    biol.  d.  St.  Petersburg,  T.  ix,  p.  531. 

(4)  Reess,  Alcoholgiirungspilze,  1870. 

(5)  Schmitz,  Stzber.  d.  uiederrh.  Gesell.,  4  Aug.,  1879,  Spr.-Abdr. 
p.  18. 

(6)  Brefeld,  Bot.  Unters,  über  Hefepilze,  der  Schimmelpilze,  v 
Heft,  1883,  p.  178. 

(7)  See  Thuret  et  Boruet,  Notes  algologiques,  ii,  p.  102. 

(8)  Engelmann,  Bot.  Zeitg.,  1879,  Sp.  49. 

(9)  Schmitz,  Stzber.  d.  niederrh.  Gesell.,  6  Dec,  1880,  Sep.-Abdr., 
p.  7. 

(10)  Schmitz,  die  Chromatophoren  der  Algen,  p.  9. 

(11)  See  for  example,  Falkenburg  in  Schenk's  Handbuch  der 
Botan.,  Bd.  ii,  p.  304. 

(12)  Z')pf,  Bot.  Centralbl.,  Bd.  x,  p.  32 ;  zur  Morpholog.  d.  Splatpfl., 
1882. 


LESSON  XXI. 

SCHIZOMYCETES.       USE    OF    THE    IMiMERSION    SySTEM. 

Finally,  we  will  examine  some  forms  from  the  small- 
est organizations,  the  Bacteria  (\)  in  order  to  become 
acquainted  with  the  morphological  rehitions  there  pre- 
vailing. We  will  not  at  first  select  any  particular  species 
for  investigation,  but  will  trust  to  chance  for  our  speci- 
men. Boil  some  green  leaves  of  lettuce  in  a  glass  vessel 
and  let  it  stand  open  in  a  room  of  relatively  high  temper- 
ature. In  another  glass  vessel  put  some  pease  killed  by 
steeping  in  boiling  water  and  pour  water  over  them. 
Distribute  small  fragments  of  cooked  carrots,  cabbages 
and  potatoes  about  on  watch  glasses  or  object  slides,  put- 
ting them  in  warm,  moist  places,  some  in  the  open  air 
and  some  under  glass  bells.  On  the  lettuce  decoction 
there  will  be  a  mouldy  skin  formed  after  two  days ; 
and  on  the  fragments  of  the  vegetable,  small,  whitish, 
rarely  colored,  masses  of  jelly.  By  putting  a  trace  of  this 
jelly  in  a  drop  of  water  on  the  slide  and  examining  it  with 
the  highest  possible  magnification,  we  find  a  vast  multi- 
tude of  the  very  smallest  bodies  embedded  in  the  jelly. 
They  form  bead-like  series  and  may  be  found  singly  or  in 
pairs  or  united  into  threads.  This  is  a  coccus  formed  of 
Bacterium  embedded  in  jelly  and  is  called  Zooglcea. 
The  jelly  arises  from  the  swollen  membranes  of  the  Bac~ 
terium.  These  membranes  consist,  in  the  putrefaction 
Bacterium,  of  a  peculiar  albuminous  substance,  myco- 
protein,  and  of  cellulose  in  the  Bacterium  not  producing 
putrefaction.  The  Bacteria  readily  absorb  aniline  and 
azo  coloring  matter,  and  so  we  will  stain  these  by  add- 
C214) 


MORPHOLOGY    OF   BACTERIA.  215 

ing  a  little  methyl  violet,  gentian  violet,  meth}!  blue, 
fuchsin  or  vesuvin  to  the  preparation.  Hsematoxylin 
colors  the  jelly  also,  and  we  will  use  it  only  when  we 
wish  to  bring  that  clearly  into  view.  The  gentian  violet 
stains  the  Bacteria  with  the  greatest  rapidity  and  inten- 
sity. ^^'e  see  the  Bacteria  then  very  distinctly  and  can 
form  a  judgment  as  to  their  manner  of  increase,  which  is 
apparently  by  continuous  self-division.  This  method  of 
propagation  gives  these  plants  the  name  of  the  schizomy- 
cetes  or  "  dividing  fungi,"  in  opposition  to  the  "  budding" 
of  the  yeast  plant.  Perhaps  instead  of  these  bead-like  forms 
there  are  little  rods  in  the  jelly  (see  Fig.  83,  ^,  farther 
on).  By  adding  a  solution  of  iodine  to  the  preparation, 
the  rods  appear  very  distinctly  to  be  a  combination  of 
short  joints.  The  segments  appear  to  be  much  shorter 
now  than  they  did  in  the  fresh  condition  ;  there  appear 
also  transverse  walls  which  were  quite  invisible  at  first. 

Certain  Bacteria  are  distinguished  by  the  fact  that  in 
their  spore-forming  stages  they  form  a  starch-like  sub- 
stance in  their  bodies  which  shows  the  blue  or  violet  color 
when  treated  with  iodine. 

In  the  skin  formed  on  the  surface  of  the  vegetable  de- 
coction occurs  a  form  of  Zooglcea  (see  Fig.  83  A).  In 
this  scum  on  the  liquid,  the  cell-series  become  a  superfi- 
cially developed  skin  held  together  b}'  the  jelly.  This  is 
permeated  with  fine,  wavy,  elongated  parallel  filaments 
formed  of  micrococci  or  bacilli.  The  articulation  of  the 
micrococci  and  bacilli  become  very  distinct  by  treatment 
with  iodine  solution.  In  such  culture  material  as  this 
we  shall  meet  the  swarming  stage  of  development.  We 
shall  be  especially  sure  to  find  it  in  one  or  two  da}s  in  the 
pea  water.  The  Bacterium  Avill  be  found  in  rapid  d:inc- 
ing  motion,  now  forward,  now  backward,  hastening  in  dif- 
ferent directions.     Sometimes  fine  cilia  may  be  made  out 


216  IMMERSION    LENSES. 

as  the  cause  of  this    motion,  Fig.  83,  B,  and  sometimes 
not. 

If  we  investigate  the  scum  ou  the  lettuce  decoction, 
after  some  considerable  time  we  shall  find  the  micrococci 
and  the  bacilli  in  a  spore-producing  state,  Fig.  83,  C. 
The  cell  contents  of  the  bacilhim  will  collect  at  one  or 
more  ])oints  and  produce  an  ellipsoidal  or  roundish  body, 
which  afterwards  becomes  darker  and  is  the  "  resting- 
spore."  This  is  preserved,  while  the  empty  membrane  of 
the  cell  finally  is  dissolved. 

In  other  cultures  we  frequently  find  bacilli  which  pro- 
duce resting-spores  only  in  one  end.  Such  forms  are 
peculiar  to  the  very  widely  distributed  butyric  acid  fer- 
ment, Clastridiam  batyricum.  Since  the  Bacteria  are  the 
smallest  known  organisms,  it  is  necessary  to  employ  our 
best  lenses  and  best  illumination  in  any  thorough  study  of 
them.  By  this  we  mean  the  homogeneous  immersion  ol)- 
jectives  and  the  Abl)e  illuminating  apparatus.  Still,  in 
most  cases,  water-immersion  lenses  can  be  made  to  suffice. 
These  lenses  may  be  applied  to  the  stand  already  de- 
scril)ed,  but  the  Abl)e  illluminating  apparatus  cannot;  for 
that  we  must  have  a  larger  stand. 

The  observer  who  works  with  a  water  immersion  must 
have  cover-glasses  made  of  a  definite  thickness,  corres- 
ponding to  the  correction  of  his  objective  system.* 

If  there  is  a  screw-collar  adjustment,  the  objective  can 
be  fitted  to  any  thickness  of  cover-glass  within  permissi- 
ble limits  by  means  of  the  correction  api)aratus.  On  the 
Zeiss  objectives,  the  figures  are  marked  for  each  0.01  mm. 
difference  in  the  thickness  of  the  cover-glass,  and  corres- 
pondingly on  lenses  of  other  makes.  Put  a  drop  of  dis- 
tilled water  on  the  front  lens  of  the  objective,  and  take 

*Thts  woulil  b3  nocassary  only  for  thoss  lenses  made  witlioiit  screw  collar  ad- 
justment, as  most  American  water-immersion  lenses  are  not.— A.  B.  H. 


ZEISS    MICKOSCOPE 


217 


Fig.  81.  Zeiss  stand  V,i,  §  natural  size.  The  body  may  be  incline<i  but  not  re- 
volved. Abbe  ilbiminaling  apparatus  attached,  c,  condenser;  d,  diaiihragm  car- 
rier;  t,  rack  and  pinion  for  tlie  same;  s,  double  mirror. 


218  HOMOGENEOUS    IMMERSION   LENSES. 

care  that  it  does  not  dry  out  during  the  observation,  but 
as  it  lies  between  the  lens  and  the  cover-glass  it  is  in  a 
situation  quite  unfavorable  to  evaporation  and  so  will  hold 
out  generally  for  some  hours.  One  must  also  be  care- 
ful that  in  shoving  the  object-slide  about  he  does  not  run 
the  drop  over  the  edge  of  the  cover-glass  and  mix  it  with 
the  fluid  used  in  innnersing  the  object  for  examination. 
If  this  should  happen  the  objective  should  be  immediately 
cleaned,  and  the  drop  of  fluid  on  the  cover-glass  removed. 
If  one  does  not  know  the  thickness  of  the  cover-glass  used, 
the  ^eorrection  of  the  objective  must  be  made  experimen- 
tally during  the  observation ;  this  is  done  by  turning  the 
adjusting  collar  back  and  forth  till  the  place  is  found  where 
the  image  is  the  sharpest  and  clearest.  Since  most  of 
the  objectives  are  made  so  that  in  this  manipulation  the 
front  lens  is  immovable,  the  object  remains  clearl}^  in 
focus. 

The  homogeneous-immersion  lenses  are  without  appai-a- 
tus  for  correction,  since  the  thickness  of  the  cover-glass 
within  allowable  limits  is  a  matter  of  indifference.  A  drop 
of  the  immersion  fluid  is  put  on  the  front  lens  of  the  ob- 
jective. It  may  be  cedar  wood  oil,  or  fennel  oil  with  cas- 
tor oil.  The  smallest  possiI)le  quantity  of  the  fluid  should 
be  used,  since  it  will  not  evaporate,  and  will  not  need  to 
be  renewed  duriuo-  the  observation.  Care  must  be  taken, 
as  in  the  other  case,  not  to  let  the  fluid  run  over  the  edge 
of  the  cover-glass.  A  perfectly  clean  linen  cloth  should  be 
used  for  wiping  the  objective.  A  piece  of  cloth  moistened 
with  chloroform  may  be  used  for  the  cover-glass.  The 
homogeneous  innnersion  lens  will  bear  the  use  of  the  high- 
est eyepieces. 

In  case  one  has  a  large  stand  like  Zeiss,  Va,  Fig.  81, 
and  an  Abbe  illuminating  apparatus,  the  upper  body  of 
the  stand  should  be  swuno;  back  even  farther  than  in  the 


ABBE  ILLUMINATING  APPARATUS.         211) 

illustration  in  order  to  affix  the  a})paratus.  This  is  done 
by  removing  the  common  mirror,  and  in  place  of  the  same 
putting  in  the  whole  Abbe  apparatus,  which  consists  of  a 
condenser  c,  a  diaphragm  carrier  d,  and  a  double  mirror 
5,  all  made  in  one  piece.  The  condenser  should  be  run 
back  till  the  upper  surface  comes  a  very  little  below  the 
upper  surface  of  the  stage  as  seen  in  the  figure.  As  a 
rule  use  the  plane  mirror.  The  concave  mirror  should 
be  used  only  with  low  powers  when  the  field  would  not  be 
uniformly  lighted  Avith  the  plane  mirror.  Diaphragms 
also  should  be  used,  and  the  narrowest  that  will  give  suf- 
ficient illumination.  If  it  is  desired  to  use  a  dark  field 
diaphragm,  turn  the  diaphragm  carrier  out  to  the  right 
from  under  the  stage  and  put  the  disk  in  and  then  push 
the  carrier  back  to  place.  The  rack  and  pinion,  t,  on  the 
diaphragm  carrier,  serves  to  move  the  diaphragms  out  of 
the  optical  axis  of  the  microscope,  about  which  axis  the 
whole  also  can  be  revolved  in  its  mounting.  By  this  means 
we  get  oblique  illumination,  but  to  this  one  seldom  has 
recourse. 

The  Abbe  illuminating  apparatus  is  so  convenient  in  use 
and  possesses  so  many  advantages  in  difficult  investiga- 
tions that  it  cannot  be  too  highly  commended.  If  one 
has  a  large  stand  with  this  apparatus,  he  should  use  it  at 
all  times  even  with  the  lowest  powers  ;  since,  by  the  inter- 
change and  movement  of  the  diaphragms,  we  may  get  any 
desired  modification  of  illumination. 

For  dark  weather  and  for  the  evening  one  needs  a  lamp 
with  a  large  burner,  and  then,  between  this  and  the  mirror 
of  the  microscope  a  large  globe  filled  with  a  very  dilute 
solution  of  cuprammonia  may  be  placed.  It  will  be  an  ad- 
vantage to  the  eyes  in  working  with  the  microscope  at 
night  to  have  the  surrounding  objects  very  nearly  as  bright- 
ly illuminated  as  is  the  field  of  the  microscope. 


220  STAINING   BACTERIA. 

The  coloring  substances  already  mentioned  above  for 
staining  Bacteria  should  be  prepared  in  an  aqueous  solu- 
tion and  should  be  used  fresh,  or  at  least  freshly  filtered. 
For  this  purpose  one  should  have  a  saturated  alcoholic  so- 
lution always  on  hand  and  from  this  drop  a  little  into  a 
considerable  quantity  of  distilled  water;  vesuvin,  how- 
ever, must  be  dissolved  in  water,  as  alcohol  changes  it,  but 
should  be  filtered  each  time  before  using  it.  The  Bacteria 
found  in  fluids  should  be  spread  in  as  thin  a  layer  as  possi- 
ble on  the  cover-glass  and  then  allowed  to  dry  in  the  tem- 
perature of  the  room.  If  the  fluid  contains  albuminous 
bodies  or  mucilage,  these  should  be  fixed,  after  being  per- 
fectly dried,  by  laying  the  cover-glass  for  several  days  in 
absolute  alcohol,  or  simpler  still,  by  subjecting  it  to  a  high 
temperature  by  passing  it  quickly  several  times  through  a 
gas  or  spirit  flame,  the  surface  of  the  glass  containing  the 
Bacteria  being  uppermost.  The  staining  is  done  b}^  put- 
ting a  drop  of  the  fluid  on  the  cover-glass  and  lettmg  it 
work  for  five  or  ten  minutes,  or  by  laying  the  cover-glass 
on  a  quantity  of  the  fluid  in  a  dish,  and  letting  it  swim 
therefrom  ten  to  thirty  minutes.  Warming  the  fluid  to 
30°  to  60°  C.  hastens  the  operation.  After  the  staining, 
the  cover-glass  is  rinsed  in  distilled  water  and  dried  by 
the  heat  of  the  room,  after  which,  a  drop  of  turpentine 
oil,  xylol  or  cedar  oil  is  applied,  the  cover-glass  laid  on  a 
slide  and  the  investio-ation  beo:un.  If  it  is  desired  to  make 
a  permanent  mount  of  the  preparation,  the  oil  is  removed 
and  the  preparation  mounted  in  Canada  bals-am  or  dammar 
which  has  been  dissolved  in  turpentine,  not  in  chloroform. 
If  the  preparation  is  to  be  examined  with  a  homogeneous 
inunersion,  care  should  be  taken  that  the  mounting  fluid 
does  not  extend  over  the  edge  of  the  cover-glass,  else  the 
immersion  fluid  will  come  in  contact  with  it  and  dissolve 
it  and  the  Avhole  surface  of  the  cover-glass  be  besmeared 


EXAMINATION    OF    BACTEllIA.  221 

with  it.  To  prevent  this,  a  ring  of  asphalt  varnish  may 
be  put  on  about  the  edge  of  the  cover-glass,  but  not  too 
far  over  the  edge,  by  means  of  a  camel's  hair  pencil. 

Having  stained  one  of  the  larger  forms  of  Bacteria  for 
investigation  we  may  examine  the  cell  contents  by  means 
of  our  highest  objectives.  We  shall  find  it  to  consist  of  a 
homogeneous  plasma  in  which  are  embedded  fine  or  coarse 
granules  which  apparently  consist  of  fat^  No  nucleus  ap- 
pears even  in  the  largest  forms.  The  body  of  the  Bacte- 
rium is  very  seldom  colored  in  a  living  state. 

For  the  more  definite  investigation  of  the  Bacteria  we 
will  take  the  smallest  form,  the  round 
Bacteria  of  the  pockl^'mph,  Micrococcus 
vaccinm  Colin   (2).     Take  fresh  lymph 
and  drying  it  on  the  cover-glass,  stain 
as  already  directed  with  gentian- violet, 
when    the    small,    round,    dark-colored 
Micrococcus  may  be  distinguished,  singly 
or  united  in  pairs.     If  fresh  lymph  be     \-^rS{ 
put  under  a  cover-glass  and  protected     nQ.  32.     spirociueta 
from  evaporation  for  several  hours  in  a  P^ionuus.  After  staining 

■  .  with    aniline,    the    seg- 

w^arm  room,  or  better  still,  placed  in  a  ments  show  bacuii.  x 
warm  closet  heated  to  36°  C,  rosary-  ^'^" 
formed  threads  will  appear,  and  after  a  still  longer  time 
heaps  of  the  micrococci.  These  are  also  to  be  seen  in  the 
lymph  preserved  in  glass  tubes,  where  they  are  visible  to 
the  naked  eye  as  small  flocculent  masses.  These  micro- 
cocci are  introduced  into  the  human  body  by  vaccination, 
where  they  increase  and  produce  the  so-called  kinepox, 
and  by  some  unknown  process  give  immunity  from  small- 
pox. 

In  water  Avhere  decaying  algae  are  found — chiefly  Spir- 
ogyra  and  Vaucheria — are  very  sure  to  be  found  also,  thin, 
moving,  screAvlike  forms,  Fig.  82,  flexible,  twisted  threads 


222  MORPHOLOGY    OF    BACTERIA. 

which  move  rapidly  through  the  water.  They  turn  upon 
their  axes  and  bend  here  aud  there  at  the  same  time,  some- 
times suddenly  stopping  and  then  hasten  on  again.  These 
organisms  are  in  all  probability  Spirochcete  plicalilis  and 
belong  to  the  swamp  Spirochmte.  When  dried  and  stained 
as  described  above,  it  Avill  be  seen  to  be,  not  nnicellular 
but  to  consist  of  successive  joints,  shorter  or  longer  ac- 
cording to  circumstances. 

On  the  same  decaying  algre,  or  on  parts  of  other  decay- 
ing water  plants,  or  on  other  corresponding  substrata,  one 
frequently  finds  growmg  tine  threads  which  belong  to  the 
Beggiaioa  alba  (3).  These  Bacteria  are  particularly  dis- 
tributed in  water  which  receives  the  waste  from  factories, 
and  in  sulphur  hot  springs.  They  spread  over  the  masses 
of  mud  at  the  bottom  a  dirty  whitish  covermg.  They  be- 
long to  the  largest  Bacteria  and  may  be  examined  with  a 
relatively  low  power.  The  threads  are  of  diflerent  thick- 
ness (from  0.001-0.005  mm.),  are  attached  or  free,  the 
free  ones  being  but  parts  of  the  attached.  The  threads 
are  jointed,  and  the  cell  contents  distinguished  by  strongly 
refracting  granules.  If  we  dry  the  preparation  and  then 
add  sulphate  of  carbon  the  granules  will  be  dissolved. 
They  consist  of  sulphur.  In  filaments  containing  much 
sulphur  the  articulation  is  quite  indistinct  and  can  be  seen 
only  after  staining  with  aniline  or  treatment  with  hot  sul- 
phate of  soda  or  glycerine.  In  the  hot  glycerine  the 
granules  are  partly,  and  in  the  sulphate  of  soda  entirely,  dis- 
solved. By  continuous  transverse  divisions  the  filaments 
may  fall  apart  into  micrococci  and  in  the  larger  forms  these 
may  undergo  a  division  perpendicular  to  that  of  the  cells, 
dividing  into  quarters.  Swarming  micrococci,  bacilli,  and 
spiral  forms  have  been  observed  among  the  Beggiaioa. 
The  fixed  filaments  may  be  spirally  bent  in  their  upper 
parts.     The  straight  and  the  spiral  filaments  alike  exhibit 


MORPHOLOGY   OF   PACTERIA.  223 

a  creeping  movement.  The  Beggiatoa  disintegrates  the 
sulphur  combinations  of  the  waters  Avhich  it  inhabits  and 
thus  causes  a  more  or  less  considerable  discharge  of  sul- 
phuretted hydrogen. 

Take  now  another  form  which  unites  the  micrococci,  the 
bacilli  and  the  spirilli,  and  shows  also  the  filamentous  form. 
It  is  found  in  tlie  white  layer  upon  the  teeth.  Placed  in 
a  drop  of  water  and  examined  with  the  highest  powers 
there  appear  bacilli  of  various  lengths,  spiral-shaped  Sj)ir- 
ochoeta,  apparently  unjointed  filaments,  and  small  closely 
compacted  micrococci.  Recently  it  has  been  demonstrat- 
ed (4)  that  all  these  forms  belong  to  the  developmental 
stages  of  the  same  fungus  LeptotJirix  buccalis,  Rol)in.  It 
lives  as  a  saprophyte  on  the  mucous  membrane  and  on  the 
covering  of  the  teeth,  but  may  under  favorable  conditions 
become  a  parasite,  penetrate  the  tissue  of  the  teeth  and 
produce  caries.  Iodine  solution  will  bring  into  view  the 
bacilli  which  compose  the  long  filaments,  and  the  com- 
pacted micrococci  are  clearly  resolved  into  their  elements. 
These  forms  never  fail  and  it  is  questionable  if  they  always 
belong  to  the  Lepioihrix. 

It  may  be  said  in  general  that  the  investigations  of  more 
recent  times  haveshown  thatthe  different  genera  and  species 
called  from  their  outward  form  (5)  Micrococcus,  Bacteriiun, 
Bacillus,  Vibrio,  Spirillum,  /Spirochceta,  etc.,  may  belong 
to  the  morphological  circle  of  one  and  the  same  species  (6) . 

We  shall  use  these  terms  only  to  designate  and  name 
given  developmental  forms,  the  micrococcus  globular  or 
ellipsoidal  forms,  bacillus  filaments  and  spirillum  the 
corresponding  forms.  The  short  rods  will  be  called  Bac- 
teria to  distinguish  them  from  the  longer  bacilli ;  the  sim- 
ple filaments  LeptotJirix,  and  the  branched  CladotJirix; 
the  screw  forms,  with  relatively  considerable  diameter  of 
wind  and  thickness  of  filament.  Spirilla;  or,  if  they  have 


224  BACILLUS    TUBERCULOSIS. 

sulphur  ir^:^nules,  Ophido?nonas ;  with  elongated  spiral,  Vi- 
briones;  very  thin  screw-forms  with  small  diameter  of  spi- 
ral and  slender  filament,  Spirochcßta ;  band-like  pointed 
spirals,  S])iromonads ;  flexible  spirals  whose  ends  wind 
back  upon  themselves,  Spirulina  (7). 

As  we  have  seen  in  the  examination  of  the  Chroococca- 
cece  and  Oscillatorieoe,  a  like  variety  of  forms  distinguish 
difierent  stages  of  development.  A  comparison  of  the  Bac- 
teria with  those  algas  leads  in  fact  to  the  theory  of  a  near 
relationship  of  these  organisms.  We  have  among  those 
algas  also  bead  like  forms  or  cocci,  rods,  filaments  and  spi- 
rals. In  the  characteristic  of  locomotion,  and  the  ability  to 
resist  a  high  temperature,  the  alga?  named  approach  the 
fungi  under  consideration.  The  first  plants  which  show 
themselves  in  hot  springs  are  these  lowest  alg»,  but  they 
really  do  not  resist  so  high  a  temperature  ;  as,  for  example, 
the  spores  of  the  hay  bacterium  whose  ability  to  grow  seems 
only  to  be  heightened  l)y  being  occasionally  boiled.  There 
is  a  resemblance  also  in  the  structure  of  their  cell-bodies, 
and  the  two  groups  both  lack  the  nucleus  and  the  formed 
chromatophores.  They  agree  also  in  their  method  of  veg- 
etative leproduction  which  gives  the  two  divisions  their 
names.  All  this  permits  us  to  consider  the  Sdiizomyceice  as 
colorless  algt«,  or  as  one  of  the  divisions  of  the  carbon-as- 
similating alga?  which  lack  coloring  matter,  but  which  must 
be  included  in  one  class  with  them,  the  self-dividing  plants, 
the  ScldzophylrB. 

Bacillus  tuberculosis,  the  recently  recognized  cause  of 
tuberculosis,  found  in  the  sputum  of  consumptives,  has  no 
locomotive  poAver,  is  very  small,  somewhat  sharpened  at 
the  ends,  and  has  within  four  to  six  granules  which  have 
been  considered  tobe  spores.  This  Bacillus  is  character- 
ized by  a  peculiar  behavior  in  staining  which  makes  it 
possible   to  distinguish  it  from   other  bacilli.     Spread  a 


STAINING   BACILLUS    TUBERCULOSIS.  225 

little  of  the  substance  to  be  tested  as  thinly  as  possible  on 
a  cover-glass,  letting  it  dry  by  the  heat  of  the  room  ;  then 
fix  the  existing  albumen  by  passing  the  preparation  three 
or  four  times  through  a  spirit  or  gas  flame.  Make  a  satu- 
rated aqueous  mixture  of  aniline  ])y  shaking  an  excess  of 
that  body  in  water,  and  filter  through  paper  previously 
moistened  with  distilled  water,  and  to  this  add,  drop  by 
drop,  a  saturated  alcoholic  solution  of  fuchsin  or  methyl- 
violet  till  it  begins  to  show  opalescence.  The  cover-glass 
ma}^  now  be  floated  on  this  fluid  for  a  quarter  or  half  a  day 
or  longer.  The  best  ellect  is  produced  when  the  fluid  is 
heated  to  40°  or  50°  C,  when  the  action  need  be  prolonged 
not  more  than  one-half  an  hour  or  an  hour.  Then  rinse  the 
cover-glass  in  water  and  lay  it  from  thi-ee  to  five  minutes 
in  a  solution  of  three  parts  nitric  acid  and  100  parts  alco- 
hol. This  bleaches  the  whole  preparation  with  the  ex- 
ception of  the  tubercle  bacilhis,  if  any  are  present.  The 
preparation  should  be  dehydrated  with  alcohol  and  exam- 
ined in  oil  of  cloves,  the  latter  being  removed  with  blot- 
ting paper,  and  the  preparation  mounted  in  dammar  gum 
or  Canada  balsam.  The  preparation  may  be  examined 
also  in  water.  The  stained  tubercle  bacilli  may  be  seen 
with  a  magnification  of  about  300  diameters  (9).  Many 
other  methods  have  been  proposed  for  staining  the  tubercle 
bacillus,  but  only  those  which  have  some  special  advan- 
tages will  be  mentioned  here  (10).  Let  four  grains  of 
aniline  oil  be  added  to  twenty-four  grains  40%  alcohol, 
which  holds  in  solution  sulphate  of  roseaniline  or  methyl 
violet,  BBBBB.  Dilute  the  solution  one-half  with  dis- 
tilled water.  Filter  and  let  stand,  not  too  lono-.  This 
fluid  will  stain  the  bacilli  on  the  cover-glass,  after  which 
the  preparation  should  be  carefully  washed  with  distilled 
water.  If  we  desire  to  stain  the  substances  containing  the 
bacilli  at  the  same  time  we  do  the  plants  themselves,  it 

15 


226  STAINING   BACILLUS    TUBERCULOSIS. 

must  be  clone  before  the  cover-glass  is  dried,  by  U-eating 
it  with  an  aqueous  solution  of  aniline  bine,  or  with  vesu- 
vin  or  Grenadier's  carmine.  The  tubercle  bacilli  will  then 
be  very  sharply  distingnished  from  the  other  Bacteria  ex- 
isting in  the  preparation  at  the  same  time. 

This  bacillus  may  be  stairted  with  methyl-violet  alone 
if  one  will  give  the  necessary  time  to  it  (11) .  Make  a  sec- 
tion of  the  tissne  which  has  been  hardened  with  absolute 
alcohol,  or  with  chromic  acid  and  then  absolute  alcohol, 
and  place  it  in  a  small  watch-glass,  in  a  solution  made  by 
dropping  four  or  five  drops  of  the  concentrated  solution 
of  methyl-violet  in  the  glass  full  of  water.  The  tubercle 
bacilli  will  be  stained  in  from  twelve  to  twenty-four  hours, 
or  in  ten  to  twenty  minutes  by  warming  the  fluid  to  50°  C. 
"Wash  the  section  in  distilled  water,  lay  it  for  live  minntes 
in  absolute  alcohol,  and  fifteen  or  twenty  minutes  in  a  1% 
acetic  acid  solution  of  Bismarck-brown,  then  again  five 
minutes  in  absolute  alcohol,  then  in  oil  of  cloves  and  finally 
mount  in  Canada  balsam  which  has  no  chloroform  in  it. 
The  tubercle  bacilli  appear  then  as  short  rods  colored  an 
intense  blue  on  a  brown  background.  Other  Bacteria^  in 
case  any  exist,  have  lost  their  blue  color  and  have  become 
a  more  or  less  pronounced  brown.  The  dry  preparation 
on  the  cover-glass  is  colored  much  more  quickly ;  with  a 
strong  saturated  methyl-violet  solution  it  takes  a  quite  in- 
tense color  in  from  half  an  hour  to  an  hour  at  the  temper- 
ature of  the  room.  Then  wash  for  one  minute  in  absolute 
alcohol,  and  treat  for  five  minutes  with  a  concentrated 
Bismarck-brown  solution.  Rinse  with  water,  dr\',  and 
mount  as  before.  While  the  tubercle  bacilli  are  but  lightly 
stained  in  from  half  an  hour  to  an  hour  in  the  methyl- 
violet  solution  in  this  case,  the  other  Bacteria  are  imme- 
diately and  intensely  stained. 

Bacteria  found  in  other  fluids  may  be  double  stained. 


STAINING   BACTERIA.  227 

According  to  one  of  these  methods  (12)  the  fluid  is  spread 
upon  the  cover-glass,  dried  and  fixed  with  the  fumes  of 
osmicacid,  or  with  a  0.  5%  sohition  of  chromic  acid,  wash 
and  stain  for  half  an  hour  to  an  hour  with  0.0017^  ani- 
line green,  then  again  wash  for  twenty-four  to  forty  min- 
utes in  slightly  acidulated  -water  to  bleach  the  tissue. 
Then  add  for  some  minutes  a  weak  solution  of  picrocar- 
mine.  Again  wash  the  preparation  and  dehydrate  with 
absolute  alcohol  or  simply  with  drying  ;  finally,  when  nec- 
essary, clarify  with  oil  of  cloves  and  mount  in  Canada 
balsam. 

For  the  examination  of  Bacteria  within  the  tissue,  the 
latter  should  be  hardened  for  a  day  or  two  in  absolute  or 
in  90  to  95  %  alcohol.  Stain  with  gentian-violet  or 
methyl-violet,  and  then  bleach  the  tissue  with  strong  al- 
cohol with  a  trace  of  potash  lye  in  it.  Laying  the  prep- 
aration for  a  half  minute  in  picric  acid  produces  the  same 
result  and  at  the  same  time  gives  the  tissue  a  yellow 
tinge.  After  bleaching  the  tissue  with  alcohol  it  may  be 
again  stained  with  iodine-green,  methyl-green,  eosin,  mag- 
dala,  acidulated  fuchsin  and  other  coloring  substances  for 
which  the  Bacteria  have  no  atfiuity  (13).  A  good  double 
staining  may  be  eficcted  with  gentian- violet  and  picrocar- 
mine  (14).  For  most  cases  a  solution  of  gentian-violet  in 
aniline  w^ater  will  give  the  best  results  (15).  Prepare  the 
latter  as  directed  on  p.  225  and  dissolve  in  it  dry  gentian- 
violet  to  saturation  or  add  to  it  a  saturated  solution  of  gen- 
tian-violet in  alcohol,  five  parts  to  one  hundred  of  the  ani- 
line water.  Filter  everj^  time  it  is  used.  The  solution  may 
be  kept  for  months.  Immerse  the  section  for  some  minutes 
in  the  solution,  then  from  one  to  three  minutes  in  a  dilute 
solution  of  potassium  iodide  of  iodine  (one  part  iodine,  two 
parts  potassium  iodide,  and  three  hundred  parts  distilled 
water),  then  in  absolute  alcohol.     The  alcohol  becomes  a 


228  CULTIVATING    BACTERIA. 

piii'ple-red  and  the  section  almost  colorless.  Clarify  in  oil 
of  cloves  and  mount  in  Canada  balsam,  dissolved  in  x3^1ol. 
The  tissue  appears  colorless,  the  Bacteria  a  dark  blue. 
Certain  Bacteria^  like  the  bacilli  of  typhus  and  the  micro- 
cocci of  many  cases  of  pneumonia,  are  bleached  by  this 
process  and  so  are  thus  distinguished  from  most  other 
bacilli.  An  instructive  staining  may  be  got  with  safranin 
in  sections  hardened  in  alcohol  or  chromic  acid  (16).  Mix 
like  parts  of  saturated  solutions  of  safranin  in  water  and 
in  alcohol  and  let  the  section  lie  for  half  an  hour  in  the 
mixture,  wash  a  little  in  water  and  some  minutes  in  abso- 
lute alcohol,  then  transfer  to  oil  of  turpentine  and  mount 
in  Canada  balsam. 

For  the  examination  of  Bacteria  in  tissue  the  Abbe  illu- 
minating apparatus  may  be  used  Avith  the  greatest  advan- 
tage (17).  But  the  diaphragms  should  be  wholly  removed, 
that  the  cone  of  light  may  be  all  utilized.  By  this  means 
the  image  of  the  uncolored  part  will  almost  entirely  dis- 
appear, while  that  of  the  colored,  light-absorbing  bodies 
will  alone  remain  visible. 

After  having  learned  to  distinguish  the  various  develop- 
mental forms,  and  the  various  methods  of  investigation, 
Ave  should  next  turn  our  attention  to  the  methods  of  cultiva- 
ting the  various  Bacteria  so  as  to  be  able  to  produce  any 
desired  form  and  follow  up  its  whole  development.  For 
this  purpose  Ave  Avill  begin  Avith  dry  hay  (18)  over  Avliich 
w^e  Avill  pour  a  little  spring  Avater,  and  let  the  infusion  stand 
for  four  hours  in  a  Avarm  closet  at  a  constant  temperature 
of  36°  C.  Then  turn  olf  the  extract  Avithout  filtering  and 
dilute  to  a  specific  gravity  of  1.004.  Put  the  fluid  in  a  re- 
tort holding  500  cm.  and  stop  the  mouth  Avith  cotton.  Boil 
with  a  slight  development  of  steam  for  an  hour.  Then  keep 
it  at  a  temperature  of  36°  C.  In  the  course  of  a  day,  or  a 
day  and  a  half,  there  will  be  formed  a  delicate  gray  film 


CULTIVATING    BACTERIA.  229 

over  the  surface  of  the  water,  which  consists  of  the  zoogloea 
of  Bacterium  subtile,  the  hay  fungus  or  hay  Bacterium. 
The  characteristic  of  the  spores  of  this  Bacterium  to  resist 
a  boiling  temperature  for  a  long  time  enables  us  to  obtain 
a  pure  culture  of  it.  The  Bacteria  generally  are  distin- 
guished by  this  quality,  their  resistance  to  high  tempera- 
ture, but  the  hay  Bacterium  stands  at  the  head  in  this 
respect.  Transfer  a  portion  of  the  film  to  the  slide  and  ex- 
amine with  the  highest  powers  ;  we  shall  find  it  to  consist  of 
long,  jointed,  wavy,  parallel  threads,  which  remain  for  the 


Fig.  83.    Bacterium  subtile.    A,  film  of  mould.    X500;  B,  swarming  bacilli.   X 
1000;  C,  forming  spores.  X  800. 

most  part  in  their  position  because  they  are  held  together 
by  an  invisible  jelly.  Fig. 83,  A.  The  filaments  consist  of 
little  cylindrical  rods,  of  diiferent  lengths,  but  which  are 
generally  two  or  three  times  longer  than  broad,  the  sub- 
stance of  which  appears  homogeneous,  quite  strongly  re- 
fractive and  colorless.  The  highest  magnification  shows 
nothing:  difierent.  Chloriodide  of  zinc  colors  the  whole 
mass  of  the  bacilli  a  yellowish-brown  very  distinctly.  This 
is  the  best  of  the  iodine  solutions  to  use  for  this  pur- 
pose. The  separate  bacilli  will  appear  shorter  than  in  the 
fresh  state  but    only  because  their  terminations   are    all 


230  STUDYING    BACTERIUM    SUBTILE. 

clearly  visible.  In  order  to  make  the  bacilli  all  quite  dis- 
tinct they  may  be  stained  in  the  ways  already  described 
■with  fuchsin,  methyl-violet,  gentian-violet,  or  vesuvin,and 
fiually  mounted  for  permanent  preparations  in  Canada  bal- 
sam or  dammar.  Sulphate  of  picric  or  picrate  of  nigrosine 
may  be  advantageously  usee!  for  fixing  and  staining  the 
preparation. 

By  using  a  magnification  of  1,000  diameters  Ave  may  see 
the  dividing  process  of  the  bacilli  direct  (19).  It  is  best 
to  draw  the  portion  of  the  fihmient  under  observation,  at 
short  intervals,  by  means  of  the  camera,  for  the  purpose 
of  comparing  the  changes  that  take  place  in  the  bacillus. 
If  there  is  sufficient  nutritive  matter  in  the  fluid  used  in 
the  observation,  the  bacillus  Avill  undergo  the  process  of 
division  in  from  half  an  hour  to  an  hour  and  a  half,  and  the 
higher  the  temperature  of  the  room  the  quicker  Avill  the 
process  be.  The  bacillus  will  increase  in  length  Avithout 
diminution  in  size  till  it  reaches  a  certain  point,  then 
a  dark  diAäsion  Avail  Avill  appear  in  the  middle,  dividing 
the  two  halves  of  the  bacillus  from  each  other.  This  proc- 
ess of  division  Avill  explain  the  arrangement  of  the  bacilli 
and  filaments,  and  it  explains  also  the  undulating  course  of 
the  filaments,  the  intercalary  growth  taking  place  at  all 
points,  and  the  elongation  of  the  filament,  being  more  or 
less  hindered,  must  cause  the  lateral  sinuosity.  The  same 
cause  also  finally  produces  the  folds  in  the  film  Avhich  are 
visible  to  the  naked  eye.  We  Avill  now  transfer  a  little 
of  the  film  to  a  moist  chamber  to  l)e  examined  in  a  sus- 
pended drop.  Make  the  moist  chamber  in  the  simplest 
possible  Avay  by  cutting  from  sufficiently  thick  paper  board 
a  rim  no  Avider  than  the  slide,  and  having  the  inner  diam- 
eter of  the  same  something  less  than  tliat  of  the  coA'er- 
glass  to  be  used.  Soak  it  in  water  and  then  lay  it  on  the 
slide.     Put  a  flat  drop  of  the  culture  fluid  Avith  some  of 


EXAMINING    IN    A    SUSPENDED    DROP.  231 

the  Bacteria  on  the  cover-glass,  turn  it  quickly  over  bring- 
ing the  drop  beneath,  and  lay  it  upon  the  pasteboard  ring. 
If  the  observation  is  to  continue  long,  the  paper  will  need 
a  drop  of  water  from  time  to  time  to  keep  it  moist.  If 
the  observation  should  be  interrupted,  the  slide  must  be 
put  in  a  larger  moist  chamber  to  prevent  its  drying  up. 
If  a  definite  place  in  the  preparation  is  to  be  subsequently 
observed,  the  position  of  the  slide  may  be  outlined  on  the 
stage  of  the  microscope,  Avith  a  lead  pencil,  and  thus  the 
slide  replaced  again  in  its  original  position.  When  the  nu- 
tritive substance  in  the  fluid  is  exhausted  the  vegetative 
growth  will  cease  and  the  formation  of  spores  will  soon 
begin.  After  six  or  eight  hours,  strongly  refractive,  ellip- 
soidal spores  standing  a  little  distance  apart  will  appear. 
Fig.  83,  Ü.  The  rest  of  the  filaments  will  be  empty, 
nothing  but  colorless  envelopes  connecting  the  spores.  At 
certain  i^oints  the  spores  will  be  found  in  the  process  of 
formation,  and  Ave  shall  see  the  strongly  refractive  sub- 
stance collecting,  in  each  bacillus,  for  the  most  part  in 
the  middle.  The  collection  increases  Avhile  the  l)acillus 
finally  becomes  empty  and  the  spore  is  completed.  In 
the  course  of  a  feAV  hours  the  envelope  becomes  indistinct, 
and  after  the  lapse  of  a  day,  the  spores  are  set  free  and 
sink  to  the  bottom  of  the  drop.  The  spores  are  very 
darklv  colored  Avith  gentian  violet,  and  absorb  other  col- 
oring  matter  like,  but  more  intensely  than,  the  l)aci}li. 
The  spores  germinate  and  groAV  very  easily  when  intro- 
duced into  other  nutritive  substances — sloAvly  in  the  tem- 
perature of  the^'oom,  more  rapidly  at  30°  C.  It  is  best 
to  boil  them  for  five  minutes  and  then  gradually  cool, 
when  the  beginning  of  the  germination  Avill  be  seen  in 
two  or  three  hours  (20).  The  spore  membrane  opens  lat- 
erally and  the  new  growth  begins  at  this  point  and  slowly 
elongates  into  a  bacillus.     The  posterior  end  remains  in 


232  GROWTH   FROxM   THE    SPORE. 

the  spore  membrane.  About  twelve  hours  will  elapse 
before  the  bacillus  will  divide  the  first  time.  In  the  mean- 
time the  preparation  will  exhibit  every  stage  in  the  devel- 
opment of  the  plant.  For  the  most  part  one  will  see  the 
sprouted  bacillus  soon  set  itself  in  motion,  which  intro- 
duces the  swarmiug  stage,  in  which  condition  it  still  bears 
the  spore  membrane  with  it  on  its  posterior  end.  The 
number  of  swarming  cells  will  constantly  increase  by  suc- 
cessive self-division  till  they  fill  the  whole  fluid,  before 
they  begin  to  form  the  film.  They  finally  collect  on  the 
surface  of  the  fluid,  come  to  rest  and  produce  the  fibn. 
The  swarmei's  are  of  ditferent  lengths  and  consist  therefore 
of  a  corresponding  number  of  joints.  Fig.  83,  B.  They 
move  with  an  undulatin«;  motion.  Put  some  of  the  fluid 
on  the  cover-olass  and  stain  the  swarmers  as  directed  on 
page  220  (21).  The  swarm  spores  have  cilia  at  both  ends 
which  are  not  easily  seen  (22). 

Cultivation  of  Bacteria  is  usually  carried  on  in  small 
flasks  (23).  Many  cultures  may  be  undertaken  on  the 
object  slide  itself.  Slide,  flask  and  every  utensil  em- 
ployed should  be  sterilized.  This  is  done  by  passing  them 
rapidly  through  the  spirit  or  gas  flame,  or  before  the  be- 
ginning of  the  experiment  to  lay  them  in  absolute  alcohol 
which  quickly  evaporates  after  taking  them  out.  The 
nutritive  solution  to  be  used  in  the  culture  is  boiled  in 
the  vessel,  closed  with  a  cotton  stopper  and  covered  with 
two  thicknesses  of  blotting  paper  or  linen.  That  boiling 
for  a  single  hour  is  not  always  sufficient  for  the  extinction 
of  life  in  the  Bacteria  is  shown  in  the  case  of  the  hay  Bac- 
terium. The  adulteration  of  the  culture  usually  comes  not 
from  the  presence  of  other  spores  in  the  air,  but  from  the 
failure  perfectl}'  to  sterilize  the  utensils;  and  the  danger 
which  arises  from  an  occasional  opening  of  the  vessel,  for 
the  purpose  of   introducing   the  spores,  is  far  less  than 


BACTERIA   CULTURES.  233 

that  which  comes  from  imperfectly  sterilized  vessels  (24). 
Culture  by  the  quantity  for  the  purpose  of  obtaining  pure 
material  is  conducted  by  diiierent  methods.  1st.  The 
method  by  dividing  the  culture  (25).  This  rests  on  the 
fjict  that  if  several  of  these  forms  are  in  the  same  nutritive 
substances,  one  of  them  will  finally  prevail  over  the  others. 
When  the  culture  is  extended  so  far,  a  little  is  transferred 
to  a  second  sterilized  nutritive  substance,  and  after  a  cor- 
responding time  from  this  to  a  third  and  so  on.  Thus 
there  is  the  chance  of  getting  at  last  a  pure  culture,  and 
of  that  particular  form  which  under  the  given  conditions 
will  reproduce  itself  the  most  quickly.  2d.  The  method  of 
dilution  (26).  If  the  desired  plant  exists  in  vastly  supe- 
rior numbers  this  method  produces  mostly  very  good  re- 
sults. Dilute  a  little  of  the  fluid  containing  the  fungus 
in  pure  water,  till  there  is  probably  not  more  than  one 
plant  in  each  drop  of  fluid.  If  now  the  plant  sought  for 
be  l)y  far  the  prevailing  one,  and  a  number  of  vessels  with 
nutrient  fluid  be  provided,  and  a  single  drop  be  put  in 
each,  the  chance  is  very  great  that  in  some  of  them  there 
will  be  the  pure  culture  desired.  3d.  The  gelatine  cult- 
ure (27).  Mix  the  luitritive  fluid  with  gelatine  so  that 
at  30°  to  35°  C,  it  will  remain  fluid  but  stiffen  at  a  lower 
temperature.  For  cultures  that  require  a  temperature 
from  30°  to  40°  C,  the  agar-agar,  or  sea-moss  gelatine, 
is  to  be  preferred  as  that  does  not  soften  at  that  temper- 
ature. A  drop  of  the  nutritive  gelatine  is  spread  out  on 
the  slide  and  allowed  to  stiffen  there,  and  is  then  inocu- 
lated with  the  fungus  by  a  needle  dipped  into  the  fluid 
containing  it.  The  preparation  is  then  set  away  under  a 
water-closed  culture-glass.  The  fungus  will  propagate 
there  and  will  furnish  us  a  ready  means  of  observing  all 
the  stages  in  the  history  of  its  development,  and  give  us 
material  for  comparison  with  the  mass-culture.     A  stiflP 


234  BACTERIA    CULTURE. 

gelatine  has  recentl\'  been  made  from  the  serum  of  the 
blood  of  cattle  and  sheep  (28).  It  is  obtained  pure  for 
the  purpose  of  sterilization  in  test-tubes  stoppered  with 
cotton  and  daily  for  six  days  heated  for  an  hour  to  58° 
C,  and  then  for  several  hours  to  a  temperature  of  65° 
C,  till  the  serum  stiffens.  This  amber-yellow  transparent 
mass  shares  with  ao-ur-asfar  the  advantao-e,  that  it  can  be 
kept  at  the  incubating  temperature. 

One  niay  judge  of  the  purity  of  the  culture  in  a  mass 
by  certain  indications ;  as,  for  example,  a  uniform  turbid- 
ness,  or  a  uniformity  of  film  on  the  surface,  or  of  cloudi- 
ness at  the  bottom,  finally  a  uniformity  of  color,  or  of 
gelatinous  formation.  Likewise,  the  purity  of  a  cnlture 
may  be  assumed  when  it  is  preceded  by  an  active  fer- 
mentation or  an  intense  putrefaction  (29). 

Notes. 

(1)  For  the  statement  following  this  see  Zopf,  die  Spaltpilze;  there 
the  rest  of  tlie  literature.  For  staining  I  depend  principally  on  Hoyer, 
Gazeta  lekarska,  1884.  Appar.'itiis  for  Bacteria  culture  are,  according 
to  R.  Koch,  furnished  by  Dr.  Müncke  in  Berlin,  Louisen  Str.  58,  and 
Rundorff,  Berlin,  Louisen  Str.  47. 

(2)  Cohn,  Beitr.  d.  Biol.  Bd.  i,  p.  161 ;  Zopf,  1.  c,  p.  92. 

(3)  Engler,  Bericht  der  Commiss.  zur  Erf.  d.  deut.  Meere,  1881 ; 
Zopf,  d.  Spaltpilze,  p.  13,  75  ff.     There  also  the  literature. 

(4)  Zopf,  same  work,  p.  80. 

(5)  Cohn,  work  quoted  above  Bd.  i,  p.  125. 

(6)  See  the  literature  in  Zopf,  die  Spaltpilze,  1883. 

(7)  Zopf,  1.  c,  p.  5. 

(8)  Von  R.  Koch,  Berliner  Kleiuische  Wochenschrift,  1882,  p.  221. 

(9)  See  Friedländer,  Mikr.  Technik,  ii  Aufl.,  p.  58. 

(10)  Von  Ermengem,  Bull.  d.  seances  d.  1.  Soc.  beige  de  Microsc, 
29  Juillet,  1882,  p.  CLI. 

(11)  Baumgarten,  Zeitschr.  f.  wiss.  Mikrosk.,  Bd.  i,  pp.  53,  54,  57. 

(12)  According  to  Soubbotine,  Arch,  de  phys.  norm,  et  path.,  T. 
xm,  1881,  p.  477. 

(13)  According  to  Hoyer  1.  c. 

(14)  Weigert,  Virchow's  Archiv,  Bd.  lxxxiv,  p.  201;  Firket  in 
Bizzozero's  franz.     Uebers.  des  Manuel  de  Micro,  diu.,  p.  314. 


LITERATURE  OF  THE  LESSON.  235 

(15)  Gram,  Fortschr.  d.  Med.  1884,  p.  185. 

(16j  Victor  Babes,  Archiv  f.  Mikr.  Anat.,  Bd.  xxii,  pp.  359  und  361. 

(17)  Introduced  by  R.  Koch;  Unters,  über  Aet.  d.  Wundinfectious- 
krankheiteu,  Leipzig,  1878. 

(18)  According  to  a  method  recommended  bj^  Roberts  and  Büchner. 
See  Zopf,  die  Spaltpilze,  p.  57,  upon  which  work  I  have  generali}'  de- 
pended for  tlie  literature. 

(19)  See  Brefeld,  Schimmelpilze,  Heftiv,  p.  38. 

(20)  See  Brefeld,  p.  43. 

(21)  See  Koch  in  Cohu's  Beitrag,  z.  Biol.,  Bd.  ii,  p.  402. 

(22)  Brefeld,  p.  40. 

(23)  Büchner,  in  Naegeli's  Unters,  üb.  niedr.  Pilze,  p.  192.  There 
also  illustrations  of  the  vessels  used. 

(24)  Büchner,  Stzber.  d.  bair.  Ak.  d.  Wiss.,  1880,  p.  381,  u.  in  Nae- 
geli's Unters,  über  niedr.  Pilze,  p.  159. 

(25)  Introduced  by  Klebs,  Arch.  f.  esper.  Path.,  Bd.  i,  p.  46; 
Zopf,  p.  43  ff. 

(26)  By  Naegeli,  Stzber.  d.  kgl.  bair.  Ak.  d.  Wiss.,  18S0.  p.  410,  u. 
Unters,  über  niedr.  Pilze,  p.  13;  Buchner,  Stzber.  d.  kgl.  bair.  Ak.  d. 
Wiss.,  1880,  p.  374  and  in  Naegeli's  Unters,  über  niedr.  Pilze,  p.  146. 

(27)  Introduced  by  Brefeld.     See  Schimmelpilze,  Heft  i,  p.  15. 

(28)  Koch,  Zur  Untersuchung  pathog.  Organismen,  Mitth.  aus  dem 
kgl.  Ges,undheitsamte,  1881,  p.  18. 

(29)  According  to  Zopf,  1.  c,  p.  44. 


LESSOX  XXII. 
The  Reproduction  of  the  Alg.e.  . 

After  having  taken  a  survey  of  the  general  field  of  mor- 
phological inquiry  in  respect  to  the  higher  and  lower  forms 
of  plant  life,  it  shall  be  our  task  to  solve  by  microscopi- 
cal investigation  some  of  the  more  important  prol)lems 
involved  in  their  special  morphology.  We  shall  follow  a 
course  the  reverse  of  that  heretofore  pursued,  and  proceed 
from  the  simplest  to  the  most  highly  organized  forms. 
We  have  made  a  beginning  already  in  the  last  lesson,  with 
the  Bacteria  whose  entire  development  we  have  examined. 
We  conclude  now  with  a  consideration  of  the  sexual  and 
asexnal  processes  of  reproduction  in  the  algse. 

We  often  have  an  opportunity  to  observe  the  conjuga- 
tion of  the  Spirogyra  (1) .  The  plant  may  be  known  l)vthe 
crisp  ajjpearance  and  the  closely  attached  filaments  of  the 
mass.  The  process  may  be  easily  followed.  If  one  does  not 
wish  to  put  the  plant  on  the  slide  under  a  cover-glass,  he 
may  use  the  snspended  drop  in  the  moist-chamber,  as  de- 
scribed on  page  230,  for  studying  the  plant.  The  conju- 
gation in  most  of  the  species  takes  place  by  the  formation 
of  a  bridge,  or  connecting  passage,  between  the  cells  of 
two  filaments  lying  near  each  other.  Short  blunt  projec- 
tions appear  on  the  contiguous  sides  of  the  cells,  which 
finally  touch  and  unite  and  form  the  connecting  tube.  In 
many  cases  it  is  possible  to  distinguish  the  male  from  the 
female  filament  before  the  act  of  conjugation  by  the  swell- 
ing of  the  cells  into  a  barrel-like  shape.  After  the  con- 
junction of  the  two  lateral  processes  the  contents  of  the 

(236) 


CONJUGATION    OF   ALG^.  237 

male  cell  first  begin  to  round  themselves  up  and  separate 
themselves  from  the  cell  wall  on  all  sides ;  then  pass  into 
the  connecting  tube  and  through  the  dividing  wall  of  the 
same.  The  female  cell  has,  in  the  meantime,  gathered  its 
contents  together  upon  the  entrance  of  the  contents  of  the 
male  cell.  Both  cells  participate  in  the  contact.  Their 
contents  are  mixed.  The  chlorophyll  bands  commingle. 
The  two  nuclei  are  united  and  form  one,  but  this  can  be 
seen  only  by  straining  the  filaments  (2).  The  body  thus 
formed  soon  begins  to  contract  and  in  the  course  of  an 
hour  its  interior  cavity  has  entirely  disappeared.  It  is 
called  a  zygospore.  The  chlorophyll  bands  are  pressed 
inward  and  the  exterior  is  occupied  by  a  colorless  frothy 
protoplasm.  After  the  lapse  of  twenty-four  hours  it 
again  increases  in  size.  A  space  appears  in  the  interior 
and  the  whole  body  becomes  ellipsoidal.  The  chlorophyll 
bands  return  to  the  exterior  and  a  distinctly  outlined 
double  membrane  covers  the  spore. 

This  method  of  conjugation  is  characteristic  of  this 
whole  group  of  algte,  to  which  belong  also  besides  the 
8])irogyra  the  Zygnema  species,  so  widely  distributed 
in  fresh  water,  and  recognized  by  two  stellate  chromato- 
phores  in  each  cell ;  and  the  desmids  so  prettily  formed. 
Nearly  related  to  the  latter  are  also  the  diatoms  in  which 
occurs  the  typical  conjugation  process. 

The  Gladophova,  whose  structure  is  already  known  to  us, 
furnish  a  very  favorable  object  in  which  to  study  the  swarm- 
spores  (3).  It  is  to  be  regretted  that  it  is  not  always 
inclined  to  form  swarm-sporcs.  It  is  relatively  easy  to 
get  them  in  the  marine  forms,  by  putting  the  plant  in  a 
large  vessel  of  sea-water.  Still,  if  our  fresh-water  form, 
Clado])liora  glomerala,  when  taken  from  rapidly  flowing 
water,  be  laid  in  a  shallow  dish  with  the  water  not  over 
1  cm.  deep,  towards  evening,  the  swarm-spores  will  most 


238  SWAKM-SPORES    IN   AI^GJE. 

likely  appear  by  the  next  clay.  The  formation  of  the 
spores  begins  at  the  end  of  the  branches  and  extends  to- 
wards their  base.  Thus  it  is  easy  to  observe  all  stages 
in  their  development,  at  the  same  time.  Beginning  with 
an  unaltered  cell  at  the  base  we  look  along  toward  the 
top  of  the  branch.  We  first  notice  the  well-known  struct- 
ure and  observe  all  that  can  be  seen  without  reagents  : 
the  polygonal,  closely-aggregated  chromatophores  which 
bear  the  small,  pale  starch  grains,  and  in  part  also  the 
greater  amylum  centres ;  the  plasma  plates  which  run 
through  the  cell  cavity  and  contain  in  part  also  the  chromat- 
ophores. If,  now,  we  move  gradually  along  towards  the 
spore-forming  cells  Ave  shall  notice  first  a  change  of  color 
in  the  cell  contents.  With  a  sufficiently  high  magnifying 
power  we  shall  observe  that  the  amyliun  centres  have  dis- 
integrated into  single  starch  grains  and  the  chromatophores 
have  also  divided  into  smaller  bodies.  The  next  stage 
shows  the  chromatophores  arranged  in  a  reticulated  order 
so  that  the  colored  contents  of  the  cell  which  surrounds 
the  cell  cavity  seem  to  be  separated  into  polygonal  sec- 
tions of  nearly  the  same  size.  The  middle  of  these  sections 
seems  to  be  free  from  granules,  and  if  we  fix  and  stain  them 
we  shall  find  a  nucleus  at  that  point.  The  membrane  which 
incloses  the  whole  cell  contents  becomes  thickened  and 
easil}'  visible.  At  a  point  near  the  forward  end  of  the 
cells,  and  in  terminal  cells  quite  at  the  extremity,  a  color- 
less lenticular  mass  of  protoplasm  is  to  be  seen.  The  cell 
membrane  swells  and  is  arched  up,  at  a  point  corj-esponding 
to  the  middle  of  this  collection,  and  by  reason  of  the  swell- 
ing and  consequent  increase  of  volume  papillate  projec- 
tions appear.  The  next  change  consists  of  the  drawing  of 
the  chromatophores  toward  the  centre  of  the  polygonal 
section  and  the  consequent  separation  of  the  latter  by 
clear  boundary  lines.     This  is  followed  by  the  rounding 


SWARM-SPORES    IN   ALG.E.  239 

up  of  the  sections  and  their  separation  from  each  other. 
The  peripheral  hiyers  appear  like  protuberant  knobs.  The 
outer  membranous  layer  formed  from  colorless  protoplasm 
takes  no  part  in  this  differentiation  of  the  chlorophyll- 
bearinsj  contents  of  the  sinsjle  sections,  but  chanijes  into  a 
colorless  mucilage  which  phiys  an  important  part  in  the 
discharge  of  the  swarm-spores.  Corresponding  to  the 
thick  collection  of  colorless  protoplasm  at  the  subsequent 
place  of  exit,  is  the  mass  of  formed  mucilage,  here  the 
greatest,  and  the  still  coherent  mass  of  swarm-spores  re- 
main at  this  place  correspondingly  removed  from  the 
swelling  cell  wall.  In  the  mulberry -shaped  mass  of 
swarmers  the  cylindrical  inner  cavity  may  be  seen ;  but 
in  case  the  spores  are  very  plentifully  developed,  this  may 
be  lacking,  but  it  commonly  exists  and  the  spores  form 
two  or  three  layers  about  it.  The  spores  are  pear-shaped. 
The  forward  pointed  colorless  end  may  be  easily  distin- 
guished from  the  rounded  posterior  end  containing  chlor- 
ophyll. At  the  front  end  is  a  red  spot  called  the  "'eye 
speck."  The  cell  membrane  is  so  much  swollen  at  the 
place  where  the  papillae  are  that  its  outline  can  scarcely 
be  made  out.  By  a  continuous  observation,  one  will  see 
the  beginning  of  the  emptj'ing  out  of  the  swarm-spores. 
By  the  pressure  of  the  swelling  cell-contents  the  papillae 
will  be  ruptured,  and  the  spore  mass  will  be  thrust 
violently  out.  Likewise  the  finely  granular  contents  of 
the  cell  cavity  will  come  out  with  the  spores,  the  latter 
after  a  while  setting  themselves  in  motion.  The  con- 
tents of  the  sporangium  show  a  diminution  of  mass,  draw 
back  from  the  cell  wall,  the  gelatinous  mass  which 
pressed  upon  the  cell  contents  apparently  lying  agjunst 
the  wall.  If  any  of  the  spores  remain  in  the  sporangium 
they  soon  set  up  a  movement  among  themselves  and  one 
after  another  escapes  through  the  papillae.     Some  remain 


240  SWARM-SPOKES    IN    ALGJE. 

behind.  If  one  examines  the  object  in  a  suspended  drop, 
he  will  find  many  of  the -spores  collected  on  the  side  to- 
wards, or  opposite  to,  the  window,  under  the  influence  of 
light,  but  those  which  are  not  sensitive  to  light  continue  to 
swim  about  for  a  long  time  in  an  indetinitepath,  and  grad- 
ually, with  the  diminution  of  their  energy,  reach  the  edge 
of  the  drop,  where  they  come  to  rest.  There  they  are 
rounded  up  and  covered  with  a  cell  membrane.  The 
spores  are  very  well  fixed  with  a  little  potassium  iodide  of 
iodine,  Fig.  84.  Two  cilia  are  seen  (four  in  some  species 
of  Cladojjhora)  to  spring  from  a  projection  on  the  ante- 
rior end  of  the  spore.  If  the  spore  lies 
in  a  favorable  position  an  application  of 
the  iodine  solution  will  reveal  a  small 
nucleus  in  its  forward  colorless  end — see 
the  fiojure  —  the  nucleolus  bein«^  foi'  the 
most  part  very  distinctly  stained. 

Fig.  84.   Cladopliora 

giomerata.      Swiinn-        ihcsc  swarui-sporcs  are  asexual,  but 
spore  lixed  With  po.  ^j^^   Cltidophora  produces  other  smaller 

tassium  iodide  of  lo-  -tr  I 

dine.    On  the  right  spores  wliich  are  scxual    and  conjugate 

side  is  seen    the  eye         .   ,  ,  im       i    *j        i  4I 

speck.  Andinthecoi-  With  ouc  another.     Ihe  latter  have  thus 

orless  anterior  end  the    f.^^.  ]^QQy^  fouud  Oulv  iu  marine  phuits  (4). 
nucleus.  X  5«.  1      "    -•    o-    7 

Jb  rom  the  order  ot  oi^pnonaceoe  we  se- 
lect Vaucheria  sessilis  for  the  study  of  the  formation  of 
its  swarm-spores  and  sexual  organs.  If  we  collect  a  stout 
specimen  from  standing,  or  better  still  from  flowing,  water 
and  lay  it  in  a  flat  vessel  with  fresh  water,  we  may  quite 
confidently  reckon  on  a  number  of  swarm-spores  the  next 
morning.  They  will  be  all  the  forenoon  discharging,  so 
we  ma}'^  easily  find  all  wished-for  stages  of  development 
(5).  If  we  look  over  the  whole  plant  with  a  magnifying 
glass  we  shall  easily  recognize  the  first  beginnings  of  the 
sporangium  in  the  darker  color  of  the  ends  of  the  fila- 
ments.    When  a  group  of  the  filaments  is  fouud  which 


SWARM-SPORES    IN   VAUCHERIA. 


241 


appears  to  furnish  the  desired  condition,  it  should  be 
transferred  by  the  forceps  without  injury  from  its  place  of 
growth  to  the  object  slide,  where  the  further  development 
may  be  directly  studied.  To  obviate  any  interference 
which  the  pressure  of  the  cover-glass  might  exercise  upon 
the  processes  of  development  it  is  well  to  put  a  fragment 
of  pith  or  horse  hair  under  one  edge.     If  a  sporangium 


Fig.  85.  Vaucheria  sessitis.  A  and  B.  beginning  of  the  sporangium;  C  to  E, 
formation  of  tlie  swarm  spores  from  the  contents  of  the  sporangium.  A — E,  X 
95.  F,  a  free  swarm-spoie.  X  250.  G,  a  piece  of  the  outer  colorless  plasma  layer 
which  occupies  the  anterior  end  of  the  swarm-spore.    X  -150. 


forms  in  the  end  of  a  branch,  the  contents,  rich  in  chloro- 
phyll, gather  themselves  together  and  the  cell  swells  out 
into  a  club-shaped  form.  The  cavity  within  begins  to  nar- 
row, Fig.  85,  A,  and  soon  the  upper  part  is  separated  as 
a  spherical  vacuole.  Then  the  sporangium  is  set  otF  from 
the  rest  by  a  division  wall,  by  the  formation  of  which  the 
contents  of  the  sporangium  are  separated  from  those  of  the 

16 


242  SWARM-SPORES    IX  VAUCHERIA. 

rest  of  the  frond  leaving  a  clear  space  between,  Fig,  85, 
B.  A  clear  border  is  formed  abont  the  contents  of  the  spo- 
rangium Avhich  soon  takes  on  a  radial  structure,  E.  The 
border  consists  of  colorless  protoplasm.  The  radial  struct- 
ure arises  from  the  cell  nuclei  which  gradually  collect  here 
and  are  placed  in  this  position,  F,  G.  These  nuclei  are  to 
be  seen  only  by  the  use  of  proper  reagents  and  the  highest 
magnification  (6).  When  the  swarm-spore  is  ready,  it 
must  be  set  free.  This  is  done  in  the  following  manner. 
The  sporangium  is  torn  with  a  jerk  and  in  the  same  in- 
stant the  anterior  part  of  the  swarm-spore  springs  out 
through  the  opening  and  begins  at  the  same  time  to  rotate 
on  its  longer  axis.  Its  passage  out  through  the  opening 
occupies  somewhat  over  a  minute.  Sometimes  the  forward 
part  of  the  swarm-spore  is  twisted  off  from  the  rest  which 
remains  in  the  sporangium,  the  result  being  that  each  part 
forms  a  perfect  though  smaller  swarm-spore.  This  is  made 
possible  onl}'  by  the  fact  that  the  spore  contains  many  nu- 
clei and  so  there  is  the  one  necessary  for  each  part.  The 
movement  of  the  swarm-spore  continues  for  about  a  quar- 
ter of  an  hour,  the  direction  of  it  not  being  influenced  by 
the  light.  The  spore  is  oviform,  the  larger  end  forward, 
which  contains  the  cell  cavity.  The  ciHa,  which  cover  the 
whole  body  as  a  short  down,  are  seen  only  at  the  moment 
when  the  spore  comes  to  a  rest,  for  in  the  next  moment 
they  are  withdrawn  into  the  body  of  the  spore,  which 
shows  during  this  process  a  wrinkled  surface.  After  that, 
the  surface  becomes  smooth.  During  the  withdrawal  of 
the  cilia,  a  thin  membrane  is  seen  to  be  formed  about  the 
spore,  which  now  rounds  up,  the  colorless  border  disap- 
pearing, the  chlorophyll  grains  returning  to  the  surface 
and  the  cell  wall  becoming  rapidly  thicker. 

In  the  terrestrial  form  of  VaucJieria  sessiUs,  the  sexual 
organs  are  easily  found  ;  the  pistillate  organ,  the  oögon- 


REPRODUCTIVE    APPARATUS    IN  VAUCHERIA.  243 

niuiii,  being  attached  immediately  upon  the  thalhis  fila- 
ment, the  antheridium  on  the  other  hand  terminatinsf  a 
branch  bent  like  a  ram's  horn  and  growing  immediately 
from  the  thallus  filament.  The  two  usually  form  a  pair 
near  each  other  ;  sometimes  one  antheridinm  is  seen  placed 
between  two  oogonia.  One  should  select  plants  of  this 
species  for  ol>servation,  and  not  those  of  Vaucheria  terres- 
tris  often  found  on  moist  ground,  for  here  the  oogonium 
and  the  antheridium  are  set  on  a  common  lateral  branch. 
The  Vaucheria  sessilis,  living  in  water,  forms  in  culture  the 
swarm-spores  already  studied,  and  after  a  few  weeks  pro- 
duces sexual  organs  also. 
The  oogonium,  Fig.  86,  o, 
is  obliquely  egg-shaped, 
filled  with  plasma  contain- 
ing oil  and  chlorophyll,  and 
separated  from  the  thallus 
thread  by  a  division  wall 
somewhat  above  its  place 

.  _  ..         Fig   86.     Vaucheria  sessilis.  piece  of  the 

ot  msertion   (7).       The  OÖ-  tliallus  with  reproductive  organs,    o,  oögo- 

gonium    is  i)rovided   with  "'""V  "'"""'''"'^'""V '''• '"'™™^'°''''''''''' 

°  ^  01,  oil  drops;  n,  nucleus,  seen  only  when 

a  lateral  bill-shaped  out- p™periy  stained,  x-^w. 
growth  in  which  colorless  protoplasm  is  collected.  The 
latter  occupies  the  whole  upper  third  of  the  oogonium  in 
some  stages  of  its  development.  Bj'  a  continuous  obser- 
vation of  such  an  oogonium,  we  see  the  colorless  contents 
at  the  end  send  out  a  papillate  process  which  slowly  rounds 
out  into  a  sphere  and  separating  from  the  oogonium  slowly 
sinks  to  the  bottom  of  the  water.  This  observation  teaches 
not  that  the  membrane  at  the  end  of  the  oogonium  is  peu- 
forated,  but  rather  that  it  swells  into  a  jelly-like  envelope 
and  the  drop  of  plasma  is  pressed  out  through  the  gelatin- 
ous substance.  The  remaining  contents  of  the  ougcjnium 
round  up,  its  colorless  end  being  the  germinal  vesicle. 


244     EEPRODUCTIVE  APPARATUS  IN  VAUCHERIA. 

The  branch  bearing  the  antheridium  is  more  or  less  bent, 
its  upper  third  is  set  off  from  the  rest  by  a  division  wall 
and  becomes  the  antheridium,  Fig.  86,  a.  In  its  ripe 
state  it  is  distinguished  by  its  colorless  contents,  while  the 
branch  which  bears  it  is  rich  in  chlorophyll  grains.  The 
apex  of  the  antheridium  is  usually  turned  away  from  the 
oogonium.  In  the  colorless  contents  of  the  antheridium, 
short  rods  longitudinally  arranged  may  be  more  or  less 
clearly  distinguished.  At  the  time  when  the  oogonium 
exudes  a  part  of  its  plasmatic  substance,  the  antheridium 
opens  at  the  apex  and  discharges  its  mucilaginous  con- 
tents. The  greater  part  of  it  remains  in  the  form  of  col- 
orless bubbles  in  the  surrounding  water  where  it  slowly 
disorganizes.  A  smaller  part  assumes  the  form  of  very 
minute  spermatozoids.  These  lively  swarming  spermato- 
zoids  soon  collect  on  the  gelatinous  mass  at  the  end  of  the 
oogonium.  Some  penetrate  to  the  colorless  embryo-sac 
of  the  spore,  and  in  favorable  cases  may  be  seen  to  com- 
mingle with  that.  After  a  short  time  the  fertilized  spore 
—  oospore  —  will  be  surrounded  by  a  delicate  membrane 
which  may  be  seen  with  special  distinctness  at  the  embryo- 
sac.  In  the  space  of  a  few  hours  the  colorless  protoplasm 
is  distributed  uniformly  through  the  oospore.  Older  spores 
are  filled  with  large  oil  drops,  show  a  brown  spot  on  the 
inside  and  possess  a  hard  cell  wall.  If  one  fixes  the  mov- 
ing spermatozoids  with  potassium  iodide  of  iodine  it  will 
be  found  to  be  provided  with  two  laterally-inserted  op- 
positely-arranged cilia  of  unequal  length. 

Notes. 

(1)  de  Bary,  Conjugaten,  p.  3;    Strasburger,  Befr.  und  Zellth.,  p. 
5;  Kny,  Wandtafln,  Text,  p.  11. 

(2)  Schmitz,  Stzber.  der  niederrli.  Gesell.,  4  Aug.,  1879,  p.  23. 

(8)  Thiu-et,   Ann.  d.    sc.  nat.  Bot.    lu  S^r.,  xiv   T.,  p.  219,   und 


LITERATURE  OF  THE  LESSON.  245 

Taf .  16 ;  Schmitz,  Siphoiiocladiaceen,  p.  34,  u.  Chromatoplioren, 
p.  119,  Anm. ;    Strasburger,  Zellb.  u.  Zelltli.,  iii  Aufl.,  p.  72. 

(4)  See  Areschoug,  Observ.  phycol.,  ii,  Acta  soc.  scient.  Upsal,  Vol. 
IX,  1874. 

(.5)  Tliuret,  Ann.  d.  sc.  nat.  Bot.,  2  ser.,  Bd.  xix,  p.  270;  Stras- 
burger, Zellb.  u.  Zellth.,  iii  Aufl.,  p.  213,  u.  84. 

(6)  Schmitz,  Stzber.  d.  uiederrh.  Gesell.,  4  Aug.,  1879,  Sep.  Abdr., 
p.  4 ;  Strasburger,  work  before  quoted,  p.  88. 

(7)  See  Priugsheim,  Monatsber.  d.  kgl.  Ak.  d.  "Wiss.  zu  Berlin  aus 
dem  Jalir  1855;  de  Bary,  Ber.  d.  Freib.  Naturf.  Gesell.,  1856;  Stras- 
burger, same  work  quoted,  p.  90. 


LESSON  XXIII. 
Keproduction  of  the  Fungi. 

If  one  puts  a  piece  of  moist  bread  under  a  glass  bell, 
in  a  few  days  it  will  be  covered  with  a  thick  mat  of  fun- 
gus filaments  which  belong  to  the  Pity  corny  ceim,  Mucor 
mucedo  (1).  It  grows  very  luxuriantly  on  fresh  dung 
kept  in  a  close  moist  place.  Its  fruiting  filaments  rise 
above  the  substratum  several  millimeters  high,  turn 
towards  the  source  of  light,  and  are  terminated  each  by  a 
round,  yellow  or  brown,  minute  bead  which  may  be  easily 
seen  with  the  magnifying  glass.  By  transferring  some 
of  the  plant  to  a  drop  of  water  on  the  slide  and  suflicient- 
ly  increasing  the  magnification,  it  may  be  demonstrated 
that  the  mycelium  consists  of  thick,  much-branched  irreg- 
ularly-divided tubes,  out  of  which  arise  these  straight 
undivided  and  unbranched  filaments  which  bear  the  spher- 
ical sporangia  at  the  top.  Those  which  are  unripe  pre- 
serve their  form  in  water  and  have  a  yellow-brownish 
protoplasm.  In  the  youngest  stages,  the  fruit  stem  is  not 
marked  oflffrom  the  sporangium,  but  further  on  a  division 
wall  arched  strongly  outward  is  produced  on  the  inside  of 
the  sporangium,  so  that  the  fruit-stem  ends  in  the  spo- 
rangium, in  a  so-called  "columella,"  a  club-shaped  proc- 
ess. The  ripe  sporangia  are  disintegrated  in  water,  only 
small  fragments  of  the  wall  remaining,  formed  of  fine 
needles',  which  consist  of  the  oxalate  of  lime  (2).  The 
freed  spores  lie  nearly  at  a  uniform  distance  apart  em- 
bedded in  a  colorless  mucilage  as  may  be  demonstrated  by 
moving  the  cover-glass.  Beneath  the  columella  is  ii  small 
collar  which  constitutes  the  remainder  of  the  calcareous 

.    (2^6) 


REPRODUCTION  OF  THE  FUNGI.  247 

incrustation.  In  the  wall-linins;  of  the  fruit-bearino-  fila- 
ment,  if  it  be  not  too  old,  one  may  follow  the  longitudi- 
nally running  streams  of  protoplasm.  Mucor  mycelium 
are  poly  nucleated,  the  nuclei  very  small  and  seen  only  by 
staining.  On  the  manure  culture  the  funsus  occasionally, 
yet  rarely,  develops  zygospores  which  appear  as  black 
points.  They  are  produced  by  the  conjugation  of  the 
club-shaped  ends  of  the  hyphte  or  m^'celium  thread.  On 
the  ripe,  dark,  warty  zygospores  one  may  see  the  two  my- 
celium threads  as  clear  circumscribed  circular  spots. 

The  cause  of  the  potato  l)light  is  a  Phy corny ceta,  the 
Phyto])litliora  infestans  (3),  whose  germinating  filaments 
penetrate  through  the  epidermal  cells  into  the  intercellular 
spaces  of  the  leaf,  and  ramifj'ing  there  destroy  the  tissue 
of  the  leaf,  forming  brown  flecks  on  the  surface  which  con- 
stantly increase  in  size.  In  order  to  obtain  the  plant  in  a 
fruiting  state  in  a  larger  mass,  put  a  blighted  branch  of  the 
plant  under  a  glass  bell,  the  air  in  which  is  saturated 
with  vapor  of  water,  and  let  it  lie  there  for  two  days. 
The  blighted  leaves  soon  become  covered,  especially  be- 
neath, with  a  white  mould,  which  is  formed  of  the  filamen- 
tous fruit-bearers,  spore-stalks,  of  the  finigus.  This  mould 
is  particularly  well  developed  on  the  edges  of  the  brown 
spots.  A  superficial  section  shows  us  that  the  spore-bear- 
ing filaments  grow  out  of  the  widely  opened  stomata.  This 
fact  may  be  observed  indeed,  though  not  so  satisfactorily, 
by  using  a  piece  of  the  leaf  of  full  thickness.  These  co- 
nidia-bearing  filaments  appear  to  .be  delicate,  unicellular 
threads  filled  with  finelj'  granular  protoplasm  and  branched 
at  top.  Fig.  87,  ^.  The  branching  is  monopodial,  and 
the  number  of  branches  but  two  or  three,  which  have  ir- 
regular swellings  along  their  course. 

The  conidia-bearing  filaments  in  dry  air  collapse  and 
twist  about  on  their  axes.     Sometimes  we  find  on  the  end 


248 


THE    POTATO    BLIGHT. 


of  the  branches  spores  in  the  process  of  development,  but 
the  ripened  citron-shaped  spores  always  fall  off  when  the 
preparation  is  put  in  water.     To  find  the  ripe  spores  in 

situ,  the  plant  must  be 
examined  dry,  but  even 
then  a  slig^ht  trace  of  wa- 
ter  should  be  introduced 
under  the  cover-glass,  for 
the  plant  rapidly  shrinks 
up  when  dry.  Specimens 
collected  in  the  open  air 
produce  the  conidia-bear- 
ing  filaments  only  on  the 
under  side  of  the  leaf. 
The  filaments  are  not  so 
long  as  those  produced 
in  the  moist  chamber  and 
therefore  not  so  easily 
seen  with  the  naked  eye. 
A  cross-section  through 
the  leaf  at  the  Ijorder  of 
the  fleck,  made  by  means 
of  elder-pith,  will  enable 
us  to  folloAv  the  course  of 
the  filament    in    its  exit 

Fig.  87.    Superficial  section  of  the  epider-  i     ^i         +                  XT  • 

mis  or  leaf  of  Solanum  hiberomm,  out  of  the  thrOUgll  the  StOUia.      r  VG- 

etomata  of  which  are  growing  the  coniiiia-  riUPUtlv           llsO        SCVCral 

bearing  filaments  of  Phytophthora  infestans.  ■' 

XöO.    iJ,  ripe  conidia;  Cone  with  the  con-  hypllPB    will     COllcct    and 

tents  divided;   A  a  swarm-spore.    B-D,  X  y^^,,^^^^^    ^^  ^^^^^    pl.^^g  and 

send  up  a  number  of 
spore-bearing  filaments.  By  following  out  the  course  of 
the  hyphffi  in  the  tissue  of  the  leaf,  we  shall  find  that  it 
runs  in  the  intercellular  spaces.  Phytophthora  is  distin- 
guished from  the  nearly  related  Peranospora  species  by 


THE    POTATO    BLIGHT.  249 

forming  but  few  and  short  processes  for  absorbing  the 
juices,  among  the  cells  of  the  host  plant  so  that  one  often 
looks  in  vain  for  them.  The  delicate  mycelium  threads, 
on  the  contrary,  cling  fast  to  the  cells  of  the  host.  The 
chlorophyll  grains  of  such  cells  first  become  brown  and 
then  they  with  the  other  elements  of  the  cell  contents  dis- 
solve and  miufjle  and  run  toijether  in  a  brown  mass,  and 
finally  the  Avhole  cell  collapses.  The  spores  are  citron- 
shaped.  Fig.  87,  B,  somewkat  pointed,  with  short  stems 
and  finel}''  granular  contents.  The  membrane  at  the  apex 
is  very  delicate  and  a  little  swollen.  The  spores  are  pro- 
duced as  we  have  seen  on  the  ends  of  the  branches  of  the 
conidia-bearing  filaments,  but  when  they  are  fully  grown, 
the  end  of  the  branch  grows  out  beyond  the  spore,  presses 
it  over  to  one  side  so  that  it  stands  nearlv  at  right  ano:les 
with  the  stem  and  finally  at  the  end  produces  a  new  spore. 
See  Fig.  87,  A.  By  sowing  the  spores  in  a  drop  of 
water  on  a  cover-glass,  and  being  careful  to  get  the  spores 
immersed  in  the  water,  and  suspending  the  drop  by  laying 
the  cover-glass  on  a  small  moist  chamber,  in  a  shaded 
place,  we  shall  have,  in  the  course  of  an  hour  or  uKn'e, 
the  beginnings  of  the  swarm-spore  forming  process. 
Since  the  swarm-spores  are  formed  from  the  contents  of 
these  larger  forms  we  call  them  conidia  and  not  spores. 
Among  the  conidia  are  sporangia  Avhich  behave  like  com- 
mon spores,  for  we  see  some  on  the  edge  or  surface  of 
the  drop  of  water  which  put  out  a  germinating  tube  from 
the  forward  papilla.  In  the  immersed  spores  the  contents 
are  divided  into  an  indefinite  number  of  cells,  (7,  which 
show  in  each  a  small  central  vacuole.  The  apex  of  the 
conidium  soon  swells  and  finally  dissolves  leaving  a  small 
orifice  through  which  the  masses  of  differentiated  contents 
are  pressed  out  one  after  another.  They  speedily  become 
swarm-spores.      By  fixing  the  swarm-spores  with  iodine 


250        SEXUAL  REPEODUCTION  OF  FUNGI. 

solution  we  recognize  tAvo  cilia  inserted  laternlly  on  the 
spoi'e  in  the  neighborhood  of  the  vacuole,  D.  The  swarm- 
spore  continues  to  move  for  half  an  hour.  It  then  comes 
to  rest,  surrounds  itself  with  a  celhilose  membrane  and 
soon  puts  out  a  germinating  tube.  This  germinating  tube 
from  a  swarm-spore  or  from  a  conidiura  direct  is  what 
penetrates  the  epidermis  of  the  stem  or  leaf  of  the  potato, 
and  so  infects  a  perfectly  sound  plant.  By  the  formation 
of  conidia  the  rapid  increase  of  the  fungus  is  provided  for. 

Sexual  reproductive  organs  have  not  yet  been  discov- 
ered in  this  species,  though  they  are  well  known  in  the 
nearest  related  Peranonpora  species.  Branches  of  my- 
celium swell  mostly  at  the  ends,  forming  a  spherical  mass 
within  the  tissue  of  the  host  plant;  which  is  separated 
from  the  mycelium  filament  by  a  division  wall.  It  is  called 
the  oogonium.  On  each  oogonium  there  lies  the  end  of 
a  mycelium  branch,  which  has  been  differentiated  as  an 
antheridium.  The  greater  part  of  the  protoplasm  of  the 
oogonium  collects  into  a  central  spherical  egg^  into  which 
the  antheridium  thrusts  a  fertilizing  tube,  whereupon  it 
surrounds  itself  with  a  thick  membrane. 

Upon  almost  any  moist  object,  which  has  the  least  trace 
of  nourishment  in  it  for  the  fungus,  may  be  found  the  blue 
green  mould,  Penicillium  crustaceum  Fries  (4).  It  is 
the  most  widely  distributed  of  all  the  moulds  and  may  be 
found  in  all  sorts  of  places.  As  convenient  a  way  as  any 
to  obtain  specimens  for  examination  is  to  moisten  a  piece 
of  bread  and  put  it  under  a  glass  ])ell,  Mucor  will  first 
appear,  but  will  be  gradually  displaced  by  Penicillium 
which  will  spread  a  blue-green  cover  over  the  substratum 
in  about  eight  days.  The  color  comes  from  the  spores  but 
o\\\y  when  they  occur  in  large  quantities.  Examine  a  lit- 
tle of  the  material  in  water.  The  mycelium  consists  of 
branched   multicellular    hypha3,    the    cells    separated   by 


SPORE   FORMATION   IN   FUXGI. 


251 


transverse  walls.  The  immediately  visible  contents  are 
finely  granular  protoplasm  witli  small  vacuoles.  Single 
filaments  not  distinguishable 
from  other  m^^celium  filaments 
have formedfruit-bearers.  On 
their  tips  is  a  whorl  of  short 
branches,  Fig.  88,  s',  which 
either  themselves  bear  whoils 
of  basidia,  or  whorls  of  short 
lateral  branches  which  do  bear 
the  basidia.  This  manner  of 
branching  gives  to  the  fruitinoj 
filament  the  appearance  of  a 
hair  pencil.  Frequently  also 
secondary  pencils  spring  from 
beneath  a  division  wall  of  the 
primary  filament.  See  the  fig- 
ure. By  a  sufiiciently  high 
magnification  we  shall  dis- 
cover that  the  basidia  are  cyl- 
indrical, prolonged  at  the  end 
into  a  finely  pointed  process, 
called  the  sterigma,  6'^.  This 
sterigma  swells  and  rounds 
at  the  end  forming  a  rapidly 
growing  spore.  Beneath  this 
is  a  second  swellin»  which 
forms  a  second  spore  and  so 
the  chain  of  spores  is  pro- 
duced. The  terminal  spores 
are  thrown  off*  Avhile  those  be- 
low are  being  produced.  PenidlUum  tufts  fixed  with  abso- 
lute alcohol  may  be  easily  colored  with  hematoxylin,  after 
which  it  will  be  seen  that  in  the  cells  of  the  mycelium  and 


Fig.  88.  Penicillium  crustaceum. 
Fruit  bearer  with  brancli  wliorls,  s' 
and  s";  basidia,  6;  sterigma,  st.  and 
spores.  Nuclei  visible.  From  an  al- 
cohol-hematoxj^lin  preparation.  X  510. 


252  SPORE    FORMING    IN   FUNGI. 

of  the  spore-bearing  filaments,  numerous  nuclei  occur  (5) . 
They  are  so  small  as  to  require  the  highest  magnification. 
They  are  elongated  in  the  direction  of  the  longer  axis  of 
the  cell  and  connected  by  fine  plasma  strings.  In  the  long 
cells  tliere  are  several,  in  the  short  cells  of  the  whorl  on 
the  aerial  filament  but  one  or  two,  in  the  basidia  but  one 
at  the  upper  end.  But  the  basidia  are  commonly  so  filled 
with  contents  at  their  apex  that  it  is  almost  impossible  to 
make  them  out.  With  the  strono;est  maofnification  one 
may  detect  a  nucleus  in  each  of  the  spores. 

Other  fruit  bodies  than  these  under  consideration  have 
been  observed  in  PenicilUum.  They  are  produced  in  cer- 
tain cultures,  have  the  size  of  a  small  pin-head,  and  are 
of  a  yellowish  color.  After  a  long  resting  period,  they 
form  asci  within,  each  of  which  produces  eight  spores. 
This  places  the  PenicilUum  among  the  Ascomycetm ,  and 
indeed  as  the  representative  of  that  division  of  the  Cleis- 
tocarp  Ascomycetoe  with  closed  fruit  bodies.  From  the 
spores  produced  in  the  asci,  the  pencil-like  fruit-bearing 
filaments  may  be  cultivated  on  the  ol^ject-slide. 

Notes. 

(1)  Brefekl,    Schimmelpilze,  Heft  i,  p.  10.      There  also  the  litera- 
ture. 

(2)  Brefekl,  1.  c,  p.  18. 

(3)  See  de  Baiy,  Aim.  de.  sc.  nat.  Bot.,  iv  s6r.,  p.  32,  und  Bei- 
träge zur  Morphl.  u.  Phys.  der  Pilze,  Heft  ii,  p.  35. 

(4)  Brefekl,  Schimmelpilze,  Heft  ii. 

(5)  Strasburger,  Zeilbikl.  u.  Zellth.,  iii  Aufl.,  p.  221. 

(6)  Brefekl,  1.  c,  p.  39. 


LESSON  XXIV. 
"Eeproduction  of  the  Fungi  and  Lichens. 

In  the  months  of  May  and  June  one  may  frequently  find 
on  the  underside  of  the  leaves  of  the  barberry,  Berberis 
vulgaris,  orange-colored  warts  which  appear  to  the  naked 
eye  to  be  finely  punctured.  A  magnifying  glass  will  show 
that  the  pillow-like  swellings  are  surmounted  by  minute 
orange-red  cups.  The  corresponding  place  on  the  upper 
side  of  the  leaf  is  marked  by  a  reddish  fleck  bordered  with 
yellow.  The  magnifying  glass  shows  it  to  contain  in  the 
inner  parts  numerous  brown  points  bordered  with  orange- 
red,  similar  points  being  found  on  the  edges  of  the  swell- 
ing on  the  underside  of  the  leaf.  The  little  cups  are  the 
aecidium  fruit  of  ^cidium  berberidis,  the  spermagonia  of 
which  are  the  above-mentioned  dark  points.  Both  together 
form  the  first  generation  of  our  common  rust-fungus,  be- 
longing to  the  JEcidioinyceteoß  or  Uridineoe,  Puccinia 
graminis,  which  completes  its  second  generation  on  our 
corn  and  other  Graminece,  producing  there  the  rust  dis- 
ease (1).  Prepare  a  delicate  section  of  the  leaf  through 
the  swellinfi:  and  examine  it  with  first  lower  and  then  higher 
powers.  We  assume  that  the  material  is  fresh,  though 
good  alcohol  material  will  answer.  Treating  with  potash 
lye  will  satisfactorily  clarify  the  fresh  section.  The  cell- 
layers  of  the  healthy  part  of  the  barberry  leaf  are  as  fol- 
lows— the  upper  epidermis,  a  single  layer  of  elongated 
palisade  parenchyma,  a  layer  of  loose  sponge-parenchyma 
about  five  cells  high,  the  lower  epidermis.  The  tissue  of 
the  affected  place  is  about  twice  the  thickness  of  the  leaf. 
Upon  the  palisade  cells  which  are   higher  but  otherwise 

(253) 


254  .    SPORE    FORMING    IN   FUNGI. 

little  changed,  is  joined  a  close  tissue  more  or  less  elon- 
gated in  a  direction  perpendicular  to  the  surface  of  the 
leaf,  and  is  distinguished  from  the  adjoining  sponge  paren- 
chyma by  its  lack  of  intercellular  spaces.  The  epidermis 
of  neither  surface  has  been  chano-ed.  The  cell  contents 
of  all  these  cells  have  undergone  disorganization,  and  con- 
sist in  part  of  colorless  drops  of  oil,  in  part  of  greenish- 
yellow  and  reddish  drops  arising  from  the  chlorophyll 
grains  and  cell-plasma,  and  of  granular  masses.  The  whole 
tissue  of  the  affected  part  shows  its  intercellular  spaces 
penetrated  by  delicate  hj'phte,  occasionally  branched,  ar- 
ticulated by  divisicm  walls  and  containing  drops  of  oil. 
They  extend  to  the  epidermis  on  both  sides.  With  chlor- 
iodide  of  zinc  and  also  with  iodine  and  sulphuric  acid,  the 
blue  color  is  not  induced  in  them,  as  fungus-celUilose  rarely 
shows  that  reaction.  Our  section  of  the  little  cups  shows 
them  to  be  more  than  half  embedded  in  the  tissue  of  the 
swelling.  We  may  easily  see  that  the  mycelium  forms  a 
thick  layer  under  the  little  cu[)s  out  of  which  arise  num))er- 
less  club-shaped  hyphpe,  perpendicular  to  the  layer  and 
parallel  to  each  other,  solidly  packed  together  and  forming 
the  so-called  hymenium.  These  hyphae,  the  basidia,  are 
transformed  at  their  ends  into  a  straight  series  of  spores, 
which  though  in  the  basidia  colorless,  and  by  mutual  press- 
ure polygonal,  gradually  round  out  and  become  orange- 
red.  The  spores  separate  from  each  other  higher  up  and 
are  discharged  from  the  opened  fruit  vessel.  An  examina- 
tion of  the  youngest  spores  on  the  basidia  teaches  us  beyond 
doubt,  that  they  are  successively  separated  from  the  point 
of  the  growing  basidia  by  means  of  a  transverse  wall. 
The  single  layer  which  constitutes  the  wall  of  the  perid- 
ium,  or  fruit-cup,  consists  of  cells  which  look  like  spores 
but  which  remain  polygonal  and  adhere  together  laterally. 
Their  fine,  delicate,  porous  walls  are  much  thickened  on  the 


SPORE    FORMING   IN   FUNGI.  255 

outside.  The  growing  peridiiim  presses  through  the  sur- 
rounding tissue  of  the  leaf,  tears  open  the  epidermis,  and 
so  comes  forth.  The  pear-shaped  spermagonia,  mainly  em- 
bedded in  the  upper  side  of  the  leaf  are  like  the  teeidia- 
spores,  surrounded  l)}'  a  thick  plexus  of  hyphjie,  from  which 
spring  closely  compressed  parallel  tln-eads  which  run  toAvard 
the  middle  of  the  organ.  These  filaments  are  very  slender, 
and  those  found  on  the  upper  part  form  a  delicate  bundle 
which  protrudes  from  the  organ.  These  threads  are  called 
the  sterigma,  are  transformed  at  their  tips  without  into  small 
globular  cells,  the  spermatia,  which  are  discharged  from 
the  organ  as  a  shiny  mass.  The  sterigma  themselves  bear 
orange-red  oil  drops,  which  lend  their  color  to  the  w^hole 
body  of  the  organ,  particularly  to  the  outside.  The  sper- 
matia do  not  germinate.  Their  significance  is  unknown. 
One  might  be  inclined  to  consider  them  the  product  of  the 
male  organ  and  to  suppose  that  a  generative  act  introduced 
the  formation  of  the  fBcidium  fruit.  As  already  mentioned, 
this  fungus  lives  in  a  second  generation  on  the  Graminem. 
It  belongs  to  the  "heterecious"  parasites,  which  in  opposi- 
tion to  the  "autoecious"  complete  their  circuit  of  life  on 
diflerent  hosts.  The  proof  of  this  is  obtained  by  sowing 
the  8ecidia  spores  on  the  germinating  plants  of  cereals  (2). 
The  uredo  growth  of  Puccinia  gramlms  meets  us  only 
too  often  in  nature  from  the  middle  of  June  till  fall,  on 
rye,  wheat,  barley,  oats,  and  particularly  on  couch-grass, 
Trilicum  repens.  It  attacks  principally  the  stem  and 
leaf  sheath  of  the  infected  plant.  One  recognizes  it  ea- 
sily as  the  slender,  rusty-brown  colored  stripes,  parallel 
to  the  nerves  of  the  leaf,  several  centimeters  long.  The 
epidermis  of  the  host  will  be  seen  torn  and  lifted  up  by  the 
underlying  layer  of  spores.  First  appears  the  rust-colored 
layer  of  uredo  spores,  with  which  are  gradually  associated 
the  brown  teleutospores.     Gradually  the  uredo  spores  are 


256  GROWTH   OF    WHEAT-RUST. 

changed,  at  last  fully,  till  the  layer  becomes  dark,  almost 
black,  and  towards  the  end  of  summer  only  teleutospores 
are  to  be  found.  If  fresh  material  is  not  at  hand,  alcohol 
material,  even  dry  plants,  will  serve  for  examination. 
Make  a  transection  of  the  stem  of  an  infected  plant.  We 
may  easily  demonstrate  that  the  hyph^e  permeate  only  a 
definite  tissue  of  the  part.  It  is  the  loose  chlorophyll- 
conta?ning  tissue  stripe,  which  alternates  with  sclerenchy- 
matous  thickened  stripe  in  the  stem,  and  which  is  covered 
with  an  epidermis  that  is  provided  with  stomata.  Here 
the  cells  are  thickly  interwoven  with  the  jointed  hypbaä 
and  their  contents  disorganized.  At  those  points  Avhere 
the  section  cuts  a  layer  of  spores  one  sees  the  mycelium 
with  many  short  and  delicate  branches  spring  up  towards 
the  surface,  which  are  headed  off  at  their  swollen  ends 
into  a  unicellular  spore,  the  uredospore.  The  epidermis 
is  cracked  open  and  its  edges  laterally  raised  up.  The 
spores  are  in  difierent  stages  of  development.  The  ri- 
pened ones  are  a  longish  oval  and  with  a  sufficiently  strong 
magnification  two  layers  may  be  seen  in  the  envelope. 
The  outer  dark  brown  is  beset  with  numerous  small  warts  ; 
the  inner  and  less  dark  shows  several,  mostly  four,  pits, 
regularly  divided  at  the  equator.  The  contents  of  the  spore 
are  granular  and  the  inner  portion  a  lively  orange-red. 

A  transection  through  a  stalk  of  oats,  having  the  dark 
brown  teleutospores,  shows  us  that  the  cause  of  the  hyphae 
is  the  same  as  previously  seen.  The  teleutospores  are 
borne  on  a  someAvhat  thicker-walled  style  than  the  uredo 
spores.  They  are  bicelhilar  oval  with  the  two  large  ends 
turned  together.  The  envelope  is  dark  brown.  The  plants 
investigated  in  the  course  of  the  season  will  show  both 
kinds  of  spores. 

The  teleutospores  survive  the  winter  and  are  capable  of 
growth  the  next  spring.     Each  of  the  two  cells  puts  out 


STRUCTUKE    OF   FUNGI.  257 

a  delicate  tube,  the  so-called  promycelium  which  divides 
transversely  into  several  cells  and  from  these  issue  vari- 
ously shaped  processes  which  divide  at  their  ends  into  kid- 
ney-shaped sporidia.  These  will  infect  the  barberry  leaves  ; 
if  they  fall  upon  one  sufficiently  young,  the  germinating 
tube  penetrates  the  outer  wall  of  the  epidermal  cells  directly 
into  the  inside  of  the  leaf  of  the  host.  We  therefore  see 
that  the  infecting  of  the  leaf  does  not  altogether  depend 
upon  the  germinating  tube  entering  a  stoma. 

In  order  to  become  acquainted  with  tlie  structure  of 
the  hymenium  of  the  HijmenomyceteGe,  (3),  we  will  select 
one  of  the  numerous  species  of  toadstools  {Amanita), 
mushrooms  {PsaUio(a),  or  agarics  (Ruasula).  We  will 
take  a  Russula  because  it  possesses  one  of  the  already 
mentioned  cystides.  Upon  the  underside  of  the  cap  are 
the  radial  lamella  which  bear  the  hymenia.  Cut  a  [nece 
out  of  the  cap  parallel  to  the  course  of  the  lamella  and 
make  the  thinnest  possible  transection  of  the  whole  per- 
pendicular to  the  latter.  The  whole  section  will  resemble 
a  comb,  the  sections  of  the  lamella  forming  the  teeth. 
With  a  low  magnification,  we  shall  see  that  the  hyphfB  go 
down  from  the  cap-disk  into  the  middle  of  the  lamella, 
thence  by  repeated  lateral  ])ranching  extend  to  the  sides 
of  the  latter.  A  portion  of  these  l)ranches  swell  into  club- 
shaped  forms  and  end  blindly,  but  a  greater  part  of  them  re- 
main slender  and  form  outside  of  the  club-shaped  branches 
a  compact  layer  of  tissue,  of  roundish  articulations,  which 
is  known  as  the  sub-hymeneal  layer,  and  is  more  or  less 
sharply  difi'erentiated  from  the  inner  tissue  mass  of  the 
lamella,  the  so-called  "trama,"  or  woof.  The  club-shaped 
branches  of  the  trama  serve  to  give  the  needed  stiflness 
to  the  lamella.  The  basidia  and  the  paraphyses  spring 
from  the  sub-hymeneal  tissue,  Fig.  89.  They  are  nearly 
parallel  with  each  other  and  set  perpendicular  to  the  sides 

17 


258 


REPRODUCTION   OF   FUNGI, 


of  the  lamella,  forming  the  hymenium.  The  basidia,  6, 
are  club-shaped.  At  their  flattened  ends  are  formed  four 
slender  branches,  c,  the  sterigma,  which  swell  out  at  their 
ends  into  an  ellipsoidal  cell,  the  basidia  spore,  sp.  These 
spores,  after  attaining  their  full  size,  remain  smooth  in 
most  cases,  but  in  many  Russula  species,  have  short  spines 
on  their  surface.  See  Fig.  89.  They  are  separated  from 
the  sterigma  by  a  division  wall  and  finally  fall  ofi".  The 
spore  carries  with  it  a  small  portion  of  the  sterigma.  The 
paraphyses,^,  are  smaller  sterile  basidia.  So  far  the  toad- 


FiG.  89.     Itussula  rubra,   a  portion  from  the  hymenium.    sh,    sub-hymeneal 
layer;  h,  basidia;  s,  sterigma;  sp,  spores;  p,  paraphyse;  c,  a  cystid.  X  540. 

stools  and  mushrooms  agree  wdth  the  description  of  the 
agarics.  But  in  the  agarics  occur  a  few  cystides,  c,  between 
the  basidia  and  the  paraphyses,  which  are  as  stout  as  the 
basidia ;  their  pointed  ends  protrude  beyond  the  general 
surface  of  the  hymenium,  and  with  their  slender  base  pen- 
etrating the  sub-hymeneal  layer,  they  represent  as  direct 
branches,  the  median  elements  of  the  trama.  AH  these 
elements  named  above  are  separated  at  their  base  from  the 
hyphas  by  division  walls,  contain  finely  granular  plasma, 
and  often  single  drops  of  oil. 


HYMENIUM  OF  THE  MOREL. 


259 


In  order  to  become  acquainted  with  the  highly  devel- 
oped form  of  the  hymenium  of  the  Ascomycetem  we  will 
select  for  examination,  Morchella  esculenta.  Dried  speci- 
mens may  be  soaked  out  and  used,  but  fresh  plants  are 
naturally  to  be  preferred.  This  well-known  morel  has  an 
irregularly  egg-shaped,  stalked  fruit-body,  which  conceals 
a  simple  cavity  within,  and  whose  upper  expanded  part  is 
laid  in  deep  folds.  The  sunken  portions  are  lined  with 
hymeneal  tissue,  which  has  not 
been  developed  in  the  projecting 
ribs  between.  Make  a  section  per- 
pendicular to  the  surface  of  some 
one  of  the  depressions.  The  hy- 
menium consists  of  spore  sacs  laid 
almost  parallel  with  each  other, 
(asci)  sap  filaments  (paraphyses), 
Fig.  90.  The  spore-tubes,  «,  are 
nearly  cylindrical  and  contain  in 
their  upper  part  eight  ellipsoidal 
single-celled  spores,  closely  pressed 
together.  The  ascus  also  contains 
a  highly  refractive  epiplasm.  The 
paraphyses  are  brownish  filaments, 
articulated  with  division  walls  and 
slightly  smaller  at  the  top.  The 
upper  cell  is  the  longest.  The  filaments  are  not  as  long 
as  those  of  the  asci.  Both  elements  are  the  ends  of  the 
hyphte  of  the  closely-interwoven  superficially-extended, 
sub-hymeneal  tissue.  This  rests  on  the  loosely-built  hy- 
phoe  tissue  of  the  fruit  body.  Treating  the  section  with 
potassic  iodide  of  iodine  colors  the  epiplasm  of  the  asci 
reddish-brown.  This  is  a  characteristic  reaction  .for  epi- 
plasm and  has  recently  been  designated  the  glycogen  reac- 
tion (4).      A  characteristic  peculiarity  of  this    reaction 


Fig.  90.  A  par:  of  the  hy- 
menium of  Morchella  esculenta. 
a,  asci;  p,  paraphyse;  sh,  sub- 
liymeneal  tissue.  X  2J0. 


260  STRUCTURE    OF    LICHEN   FUNGUS. 

shoAvs  itself  by  the  application  of  heat.  To  a  section  in 
water,  stained  with  the  iodine  reagent,  add  a  little  more 
water  but  not  enough  to  remove  the  color.  Then  gradu- 
ally and  carefully  warm  it  Avithout  bringing  it  to  the  boil- 
ing point,  laying  it  over  white  paper  occasionally  to  see  if 
tlie  color  becomes  paler.  When  this  takes  place,  rapidly 
cool  the  preparation,  and  if  it  is  a  large  one  it  will  be  seen 
by  the  naked  eye  to  take  on  its  dark  color  again  (5).  By 
means  of  potassic  iodide  of  iodine,  one  may  trace  the  be- 
ginnings of  the  asci  from  some  depth  in  the  tissue  of  the 
sub-hymeneal  layer.  The  paraphyses,  the  sub-hymeneal 
layer,  and  the  tissue  of  the  inside  of  the  fruit-body  are  col- 
ored at  the  same  time  a  yellow,  or  yellow-brown  color. 

The  fungus  in  the  thallus  of  the  lichen  belongs,  with 
rare  exceptions,  to  the  jLscoviycetece.  The  Pliyscia  ciliaris 
is  rich  in  fi-uit.  The  apothecium  is  saucer-shaped  with  an 
inclosing  border  formed  from  the  thallus.  This  diminishes 
under  the  apothecium  into  a  pedicel  a  transection  of  Avhich 
shows  a  radial  structure,  Avith  a  uniform  thickness  of  rind 
layer  following  Avhich  is  a  layer  of  gonidia  around  the 
whole  circumference.  The  inside  of  the  pedicel  is  occu- 
pied by  a  loose  texture  of  hyphse. 

We  make  next  a  median  longitudinal  section  through  the 
apothecium.  This  shows  the  structure  of  the  border  of 
the  apothecium  constructed  out  of  the  tissue  of  the  thal- 
lus. The  gonidia  layer  extends  to  the  edge  of  this  bor- 
der, from  which  at  intervals  cilia-like  processes  put  out. 
The  style  Avidens  to  inclose  the  hymenium,  Avhich  rests 
on  its  central  fundamental  tissue.  The  hymenium  is 
broAvnish.  It  consists  of  a  great  number  of  long,  ex- 
tremely slender,  jointed  filaments,  the  paraphyses,  between 
Avhich,  far  less  numerous,  stand  the  club-shaped  spore- 
sacs,  the  asci.  The  latter  are  always  of  different  age, 
the  ripe  ones  having  eight  broAvn-Avalled  spores.     The 


REPRODUCTION    OF    LICHENS. 


261 


£- 


spores  are  ellipsoidal,  bicelliilar  and  at  the  boundary  of 
the  two  cells  a  little  contracted.  Both  elements  spring 
from  a  felted,  uniformly  colored,  horizontally  extended 
layer,  the  sub-hj^meneal  layer.  This  rests  upon  the  central 
tissue  of  the  style  from  which  it  is  distinguished  by  its 
brown  color  and  its  lack  of  air-filled  spaces.  While  we 
have  seen  that  the  hyphaj  of  the  thallus  are  not  colored 
blue  with  chloriodide  of  zinc,  the  hymeneal  tissue  is  colored 
a  dark-blue  by  the  application  of  a  little  potassic  iodide 
of  iodine.  The  walls  of  the  hymeneal  elements  are  formed 
out  of  a  particular  mod- 
ification of  cellulose, 
which  is  known  as  starch 
cellulose.  Examining 
the  thallus  of  this  lichen 
with  a  magnifvinof  glass 
we  shall  find  little  wart- 
like elevations  standins:  (r-^^^v*^J-,,>^i>i-.  v^'S 
here  and  there  singly  or  ßii''^^P^'^'<^'^^^^m 
in  groups.  If  we  make 
delicate  transections  in 
considerable  n  u  m  b  e  r 
through  the  thallus  we 
shall,  with  some  of  them,  hit  one  of  these  elevations  in 
such  a  way  as  to  show  a  section  like  that  represented  in 
Fig.  91.  This  is  the  spermagonium,  an  egg-shaped  form, 
sunk  in  the  thallus  and  having  an  open  pore  or  mouth,  sj). 
It  occupies  nearly  the  whole  depth  of  the  thallus,  is  sur- 
rounded on  the  sides  by  the  gonidia  layer,  and  has  within 
a  mass  of  very  delicate,  nearly  radially-arranged  fihmients 
with  short  joints,  the  sterigma  (see  the  figure).  The 
longer  axis  of  the  organ  is  occupied  with  a  cylindrical 
cavity  which  contains  short  rod-like  spermatia  which  have 


Fig.  91.  Transection  of  thallus  of  Phijscia 
cUiaris  througli  the  middle  of  a  spermago- 
nium, sp;  c,  rind  layer;  m,  pith;  ^,  gonidia 
layer  of  the  thallus.  X    90. 


262  LITERATURE  OF  THE  LESSON. 

been  separated  from  the  ends  of  the  sterigma.  These  es- 
cape through  the  opening  at  the  top  of  the  spermago- 
nimn.  In  the  CoUemacece  it  has  been  demonstrated  that 
the  function  of  the  spermatia  is  that  of  the  male  genera- 
tive product  (6).  In  other  lichens  their  function  is  still 
unknown. 

Notes. 

(1)  See  de  Biiry,  Monatsber.  d.  k.  Akad.  d.  Wiss.  in  Berlin  für  das 
Jahr  1865,  p.  15;  Kiiy,  Bot.  "Wandtafeln,  p.  68;  Frank,  die  Krankheit 
d.  Pflanz.,  p.  454. 

(2)  de  Bary,  same  work,  1866,  p.  206. 

(3)  See  de  Bary,  Morph,  u.  Pliys.  der  Pilze,  p.  112;  Goebel,  Grund- 
züge, p.  143.     In  both  the  rest  of  the  literature. 

(4)  Leo  Errera,  L'6piplasme  des  Ascomycetes,  1882.  There  also  the 
literature  relating  to  epiplasma. 

(5)  1.  c,  p.  45. 

(6)  E.  Stahl,  Beiträge  zur  Entwicklungsgeschichte  der  Flechten, 
Heft  I,  1S77. 


LESSON  XXV. 
Reproduction  of  the  Mosses. 

The  Marcliantia  polymoijjha,  already  known  to  us,  is 
most  rapidly  propagated  in  an  asexual  or  vegetative  way 
by  means  of  asexual  buds  orgemmse.  They  are  common 
in  the  Hepaticem  generally  and  appear  in  most  exquisite 
form  in  this  species.  They  are  produced  in  the  Marchan- 
tia  in  cup-shaped  receptacles  on  the  back  side  of  the  thallus. 
The  cup  has  a  beautifully  toothed  border  and  the  vivid 
green  gemnife  are  found  at  the  bottom.  A  longitudinal 
section  through  the  cup,  parallel  with  the  long  axis  of  the 
thallus,  first  narrows  and  then  pretty  suddenly  widens  out- 
ward to  the  edge.  The  tissue  forming  the  air-chambers 
continues  up  the  outside  of  the  cup  to  the  upper  half  of 
its  outer  extension.  The  base  of  the  cup  is  occupied  with 
club-shaped  papilltB  whose  membrane  is  transformed  into 
mucilage.  BetAveen  these  club-shaped  hairs  are  occasional 
bicellular  hairs  whose  upper  cells  are  divided  first  by  trans- 
verse walls  and  subsequently  by  longitudinal  wallstill  they 
at  last  attain  a  considerable  lateral  extension,  and  finally 
become  several  cell-layers  thick  in  the  middle  and  quite 
biscuit-shaped  inform  (1).  The  single-celled  styles  are 
easily  parted  leaving  the  gemnife  loose  in  the  cup,  from 
which  they  are  soon  discharged  by  means  of  the  swelling 
mucilage  which  is  produced  in  the  bottom  of  the  cup  l)y 
the  club-shaped  hairs.  The  little  notches  on  the  side  of 
the  gemm«  form  the  vegetative  points  whence  are  pro- 
duced short  papilke.  The  cells  of  the  gemmae  are  rich  in 
chlorophyll ;  still  on  both  surfaces  of  the  organ  are  found 
large  chlorophyll-free  cells,  which  keep  near  the  middle 

(263) 


264         EEPRODUCTIOX  OF  MARCHANTIA. 

but  are  otherwise  irregularly  distributed.  In  some  of  the 
border  cells  are  oil-bodies.  When  the  gemmfe  are  sown 
and  germinate,  these  chlorophyll-free  cells  develop  in  a 
day  or  two  on  the  under  side  into  root-hairs,  and  on  the 
upper  side  into  the  tissue  of  that  side   (2). 

The  sexual  reproductive  oigans  of  Marchantia  are  placed 
on  special  receptacles.  We  "will  examine  those  of  M.poly- 
morpha  (3).  The  male  and  female  receptacles  are  easily 
distinguished,  the  former  presenting  disk-like  and  the 
latter  umbrella-like  forms.     The  two  organs  are  produced 


-f 


I, 


V^ 


Fig.  92.  Mardiantia  polymorpha.  A,  optical  trail .«ection  of  a  nearly  ripe  anthe- 
ridiiim ;  ]),  parajiliyse;  B,  spermatozoids  fixed  with  a  Ifc  Solution  of  perosmic  acid. 
A  X  yO;  B  X  tiOO. 

on  different  plants.  The  receptacles  together  with  their 
styles  represent  the  transformed  branching  of  the  plants. 
By  making  a  delicate  section  through  the  pistillate  recep- 
tacles, we  see  that  its  structure  conforms  to  that  of  the 
thallus,  its  upper  surface  answering  to  that  of  the  back 
side  of  the  frond  and  the  under  side  of  the  receptacle  to 
the  under  or  ventral  side  of  the  frond,  being  provided  like 
that  with  rhizoids  and  scales.  The  antheridia,  Fig.  92,  A, 
are  sunk  in  special  cavities  in  the  open  side  of  the  male 
organ.     The  section  shows  that  each  cavity  contains  but 


ANTHERIDIUM   OF   MARCHANTIA.  265 

one  antheridium  too-ether  with  a  few  short  sinofle-celled 
paraphyses,  p.  The  cavity  closes  over  the  antheridium 
with  the  exception  of  a  narrow  canal  which  is  left  open. 
The  antheridium  is  an  oval  body  Avith  a  short  pedicel  and 
has  an  outer  membrane  of  a  single  layer  of  cells  contain- 
ing chlorophyll.  The  special  mother-cells  of  the  sper- 
matozoids  are  produced  by  successive  right-angular  cell 
divisions,  and  form  a  series  of  transverse  and  longitud- 
inal rows  in  the  nearly  ripened  antheridium  (see  the 
figure) .  Just  before  the  ripening  of  the  mother-cells  of 
the  spermutozoids  the}'^  are  rounded  out  and  separate  and 
finally  burst  the  enclosing  membrane  of  the  antheridium 
at  its  apex,  and  the  small  round  cells  escape.  If  we 
put  a  drop  of  water  on  the  top  of  one  of  these  gi'owing 
receptacles,  we  shall  see  it  spread  rapidly  over  the  whole 
surface  and  soon  become  milky-white.  A  high  magnifica- 
tion shows  it  to  be  filled  with  numberless  spermatozoid 
cells.  They  remain  for  a  short  time  at  rest  after  which 
the  cell  membrane  begins  to  swell  up  and  finally  bursts 
and  the  spermatozoids  escape  into  the  water.  The  sper- 
matozoids  are  relatively  very  small,  have  a  filiform 
body  and  two  long  cilia,  and  attached  to  their  posterior 
end  a  minute  bladder  which  they  finally  lose  during  their 
swarming.  In  order  to  see  them  distinctly,  we  may  add 
to  the  preparation  a  drop  of  a  one  per  cent  solution  of 
perosmic  acid  which  will  fix  them  most  beautifully  and 
allow  us  to  study  them  very  conveniently.  See  Fig.  92, 
B.  The  same  result  is  obtained  by  the  use  of  a  trace  of 
polassic  iodide  of  iodine. 

The  female  receptacle  forms,  as  does  the  male,  a  radially 
arranged  inflorescence  generally  consisting  of  nine  rays  be- 
tv/een  which  are  eight  rows  of  archegonia  on  the  under 
side.  This  distinguishes  it  from  the  male  organ.  Still 
this  difierence  depends  upon  the  earlier  deflection  of  the 


266 


ARCHEGONIUM   OF   MARCHANTIA. 


vegetative  point  of  the  receptacle  towards  the  underside. 
We  shall  see  by  the  use  of  the  magnifying  ghiss  that  the 
row  of  archegonia  lying  between  the  rays  is  inclosed  by  a 
common,  one-layered,  rib-like  membrane,  bordered  at 
the  edge.  By  making  a  delicate,  longitudinal  section  be- 
tween thumb  and  finger  of  a  relatively  young  receptacle, 


o> 


0^ 

/  \n 


Mb 

I   If 


Fig.  93.  Marchantia  polymorpha.  A,  young,  B,  open  arcliegonium,  after  the  for- 
mation of  the  beginning  of  tlie  germ;  k' ,  necli  canal  cells;  k" ,  ventral  canal  cells; 
o,  ovum;  pr,  perianthium.  X  540. 

we  shall  easily  find  in  some  of  them  the  female  organ,  the 
archegonium.  The  oldest  lie  near  the  border,  the  suc- 
cessively younger  nearer  the  pedicel.  The  first,  the 
ripened  ones,  show  their  necks  beyond  at  the  edge  of  the 
disk  bent  upwards ;  the  others  run  straight  downwards. 
In  an  archegonium,  which  is  nearly  ripe,  Fig.  93,  ^,  a 
short  pedicel,  a  ventral  and  a  neck  part,  may  be  distin- 


ARCHEGONIUM  OF  MARCHANTIA.  267 

guished.  The  wall  of  the  pedicel  and  the  ventral  part  is 
composed  of  one  layer  of  cells.  The  central  cell  of  the 
ventral  part  is  filled  with  an  egg,  o,  and  a  ventral  canal 
cell,  k'  h",  which  shortl}'  ])efore  ripening  is  separated  from 
the  egg.  The  nncleus  of  the  egg  is  easily  seen.  The  neck 
has  a  central  canal  running  through  it  which  is  formed  by 
four  canal-cells  whose  division  walls  have  been  absorbed. 
The  contents  of  these  four  cells  have  comminofled  and 
formed  a  continuous  strin«;.  Between  the  archeo-onia  are 
small  leaf-like  scales  originating  in  the  receptacle.  In 
many  preparations  one  will  find  the  membrane  which  cov- 
ers and  protects  the  whole  archegonium  layer.  It  consists 
of  a  single  stratum  of  cells,  is  fringed  at  the  border,  and 
its  cell  often  contains  oily  bodies.  It  is  relatively  easy  to 
see  the  opening  of  the  archegonium  directly  under  the  mi- 
croscope. 

Make  a  longitudinal  section  of  the  female  flower  which 
is  raised  but  a  little  from  the  pedicel  and  lay  it  dry  under 
a  cover  glass  upon  the  microscope.  When  a  ripe  arche- 
gonium is  found,  add  a  drop  of  water  at  the  edge  of  the 
cover-glass,  keeping  the  preparation  under  observation, 
whereupon  the  archegonium  will  almost  immediately  open. 
The  cause  of  this  opening  is  in  the  swelling  of  the  con- 
tents of  the  canal  in  the  neck.  The  canal  cells  of  the  neck 
dissolve  at  the  apex  and  their  contents  escape  followed  by 
that  of  the  ventral  canal  cell.  The  homogeneous  part  of 
these  contents  forms  a  rapidly  swelling  mucilage  which  is 

distributed  in  the  surroundin«?  water.     The  OTanular  con- 
es o 

tents  lie  in  the  water  and  gradually  disorganize.  Di- 
rectly after  the  emptying  of  the  ventral  canal  cell,  the 
central  cell  of  the  ventral  part  is  rounded  up.  See  Fig.  93, 
JB.  On  its  anterior  border,  is  often  but  not  always  found 
a  clear  spot,  a  germinal  fleck  or  embryo  sac.  The  intro- 
duction of  the  spermatozoids  into  the  canal  of  the  neck  may 


268  FERTILIZATION   OF    ARCHEGONIUM. 

be  easily  ohserved  in  this  plant.  For  this  purpose  instead 
of  pure  water,  we  must  add  to  the  preparation  a  drop  of 
water  Avhich  has  been  for  a  time  in  contact  Avith  a  ripe 
male  receptacle.  The  spermatozoids  collect  about  and  in 
the  mucilage  which  has  exuded  from  the  archegonium,  and 
one  sees  them  enter  the  neck  where  they  become  invisible. 
A  substance  is  secreted  from  the  archegonium  which  af- 
fects the  spermatozoid  as  a  chemical  irritant  and  deter- 
mines the  direction  of  its  movement ;  so,  when  it  reaches 
the  exuded  mucilage,  it  is  gradually  moved  along  in  the 
direction  of  the  opening  of  the  neck  of  the  archegonium. 
It  is  interesting  to  notice  that  the  neck  of  an  unfertilized 
archegonium  does  not  close  up,  and  that  the  archegonium 
in  such  a  condition  gradually  perishes.  But  if  water  con- 
taining spermatozoa  be  added  to  the  preparation  and 
the  egg  he  fertilized,  the  neck  is  shut  up  in  a  few  hours  by 
the  gradual  narrowing  of  it  from  above  downward.  If  the 
preparation  be  laid  by  for  twenty-four  hours,  the  existence 
of  a  cellulose  membrane  about  the  fertilized  egg  may  be 
easily  recognized,  which  gradually  thickens  in  the  next 
following  days. 

The  fertilized  archegonium,  which  one  finds  in  a  section, 
shows  a  brown  shrunken  neck,  while  the  egg  is  already 
midergoing  segmentation,  Fig.  93,  C.  About  the  base  of 
the  archegonium  there  begins  to  develop  from  the  foot  of 
the  same,  a  cup-shaped  envelope,  the  perianth,  pr,  which 
incloses  the  whole  growing^  archegonium.  In  a  longitudi- 
nal  section  of  the  receptacle,  which  has  already  expanded 
its  marginal  rays,  one  may  see  fixed  the  living-green,  fully- 
developed  archegonia,  with  their  l)road  bases,  and  their 
apexes  adorned  with  what  remains  of  the  neck  part.  From 
the  fertilized  egg,  or  ovule,  is  produced  the  sporogonlum 
which  one  sees,  finally,  in  preparations  made  from  the 
older  receptacles.     These  sporogonia  form  a  yellow-green 


ANTHERIDIA    OF   MOSSES.  269 

oviil  capsule  with  a  short  pedicel.  The  walls  of  the  cap- 
sule consist  of  one  layer  of  cells.  By  tearing  the  mem- 
brane apart  with  needles  and  examining  with  a  higher 
power,  we  observe  a  characteristic  thickening  ring  in  the 
otherwise  thin-walled  cells.  The  3'^ellow-brown  spores  are 
finely  dotted.  Between  them  are  long  slender  cells, 
pointed  at  the  ends  and  characterized  by  two  brown,  screw- 
shaped  bands  in  their  walls.  They  are  the  so-called  "ela- 
ters."  The  interior  of  the  capsules  are  filled  exclusively 
with  spores  and  elaters.  The  open  capsule  has  the  open- 
ing set  round  with  several  recurved  teeth.  The  elaters 
are  strongly  hygroscopic,  bend  back  and  forth  by  a  change 
in  the  humidity  of  the  atmosphere  and  so  help  to  scatter 
the  spores.  All  Mardiantia  do  not  have  their  reproduc- 
tive organs  upon  such  elaborately  constructed  receptacles, 
and  in  other  Hepaticeca  these  specializations  generally  are 
wanting.  On  the  contrary,  it  often  happens  that  the  pedi- 
cel of  the  sporogonium  is  considerably  elongated,  and  the 
capsule  with  the  spores  correspondingly  elevated,  which 
also  promotes  the  scattering  of  the  spores. 

For  a  study  ot  the  antheridia  of  the  true  mosses,  Musci, 
we  will  choose  Milium  Jiornum,  a  widely  distributed  plant, 
which  forms  remarkably  fine  and  numerous  male  flowers 
in  the  month  of  May,  and  at  the  same  time  offers  female 
blooms,  or  archegonia,  for  investigation.  The  former  are 
much  more  numerous  than  the  latter,  the  latter  having 
sometimes  to  be  sought  for  a  long  time.  The  male  blooms 
are  dark  green,  disk-shaped,  inclosed  in  a  rosette  of  foli- 
age leaves,  the  so-called  envelope  or  perigoneal  leaves. 
Towards  the  middle,  the  leaves  of  the  bloom  rapidly  di- 
minish in  size.  In  the  axils  of  the  outer,  but  also  still 
more  in  that  of  the  inner  leaves,  are  found  numerous  an- 
theridia and  paraphyses  which  extend  over  the  whole  apex 
of  the  stem.     This  ma}"  be  easily  seen  in  a  longitudinal 


270  AXTHERIDIA    OF   MOSSES. 

section  of  the  bloom,  the  section  being  made  between  the 
fingers,  and  the  apex  of  the  stem  being  tnrned  downward 
in  making  it.  This  section  shows  that  the  stem  is  widened 
at  the  top  and  a  little  hollowed  out  also  where  the  repro- 
ductive organs  are  inserted.  The  central  conducting  bun- 
die,  peculiar  to  the  Mnium  species,  is  also  correspondingly 
widened  and  ends  in  a  chlorophyll-containing  tissue  which 
is  spread  out  under  the  bottom  of  the  blossom.  The  anthe- 
ridia  and  the  paraphyses  are  easily  made  out  and  their 
structure  ascertained.  The  antheridia  are  club-shaped  bod- 
ies, somewhat  contracted  at  the  ends  and  borne  on  short 
pedicels.  The  cells  of  their  one-layered  walls  contain  nu- 
merous chlorophyll  grains.  The  contents  consist  of  small 
colorless  cells  whose  division  walls  in  the  young  state  of  the 
antheridium  are  clearly  placed  at  right  angles.  If  older  an- 
theritlia  are  cut  by  the  section,  the  exuding  contents  are  seen 
to  consist  of  rounded,  ;idhcting  cells,  the  spermatozoid  cells 
in  which  the  filamentous  bodies  of  the  spermatozoids  may 
often  be  recognized.  The  chlorophyll  grains  at  the  apex 
of  the  ripe  antheridia  are  l)rownish.  Empty  antheridia  are 
opened  at  their  apex.  The  paraphyses  are  simple  cell  fila- 
ments which  gradually  expand  upward  but  again  contract  so 
that  the  uppermost  cell  is  always  sharp.  The  walls  of  the 
cells  are  often  brown  at  the  base  of  the  paraphyses  and  some- 
times higher  up  also.  They  bear  chlorophyll.  Cross- 
sections  made  through  the  under  part  of  the  bloom  show 
in  an  instructive  way  the  distribution  of  the  antheridia, 
their  relations  to  the  enveloping  leaves  and  to  the  para- 
physes, also  many  transections  of  the  antheridia  them- 
selves. 

Still  more  satisfactory  than  the  male  blooms  of  the  Mni- 
um are  the  red-colored  ones  of  the  PolytricJium  species 
wdiich  may  be  likewise  found  in  the  month  of  May.  Se- 
lect Polytrichuin  juniperinum.    The  perigoneal  leaves  dif- 


ARCHEGONIUM    OF   MOSSES.  271 

fcr  from  the  foliage  leaves  not  only  in  color  l)iit  also  in 
the  fact  that  the  single-layered  sheath-part  extends  quite  to 
the  apex  of  the  leaf.  The  formation  of  the  green  lamellae, 
characteristic  of  the  Polyirichum,  is  limited  to  the  nerve 
on  the  upper  side  of  the  leaf.  On  the  red-brown  perigoneal 
leaves  which  occupy  the  inside  of  the  bloom  and  become 
rapidly  smaller,  are  the  green  lamellee  produced,  and  only 
on  the  outermost  leaves  at  the  point  which  is  bent  sharply 
outward.  So  the  leaf  appears  finally  to  be  reduced  al- 
most to  its  sheath  part.  The  antheridia  and  the  paraphy- 
ses  stand  in  the  axils  of  the  perigoneal  leaves.  The  middle 
of  the  bloom  is  occupied  with  a  vegetative  bud  which  is 
a  continuation  of  the  central  string  of  the  stem.  In  Poly- 
irichum norynale,  the  male  flower  grows  up  through  this. 
The  antheridia  have  the  same  structure  as  in  Mnium.  The 
paraphyses  form  a  long  cell-filament  at  their  under  part, 
widen  spatulate  at  top  into  a  single  cell  layer.  If  one 
presses  a  bloom  of  the  Polytrichum  between  the  fingers,  the 
contents  of  the  antheridia  will  exude  as  a  milky  slime 
clearly  visible  on  the  red-brown  leaves. 

The  female  blooms  of  Mnium  Jiornum  are  generally  not 
so  easily  seen  as  the  male  and  one  must  hunt  for  them. 
The  plant  which  bears  them  is  much  shorter  and  has  darker 
leaves.  The  upper  leaves  close  bud-like  about  the  pis- 
tillate organ,  the  archegonium,  to  protect  it.  The  median 
longitudinal  section  shows  that  the  stem  is  not  essentially 
widened  at  the  top  but  is  bhmted  in  a  peculiar  way.  This 
may  be  a  sure  indication  to  us  that  we  have  to  do  with  a 
female  bloom  even  when  we  cannot  find  the  archegonia. 
The  central  conducting-bundle  of  the  stem  is  somewhat 
enlaroed  under  the  bottom  of  the  bloom  and  ends,  as  in  the 
male  flower,  in  a  tissue  containing  chlorophyll.  The  perie- 
chsetal  leaves  while  remainino;  foliaceous  in  form  diminish 
in  size  towards  the  middle  of  the  bloom.     In  hermaphro- 


272  ARCHEGONIUM   OF    MOSSES. 

dite  flowers  they  form  what  is  called  the  perigonium. 
There  are  but  few  archegonia  in  the  apex  of  the  flower 
so  there  must  be  an  exactly  median  section  in  order  to 
And  them.  The  archegonia  are  in  the  main  formed  on 
the  same  plan  as  those  of  the  Hepaticeoe,  only  that  the 
pedicel  is  much  more  strongl}^  developed  and  but  little  di- 
minished below,  forming  the  principal  mass  on  the  under 
half  of  the  archegonium.  The  ovum  appears  relatively 
small  on  this  foundation.  It  must  be  sought  for  imme- 
diately under  the  beginning  of  the  neck  which  is  here 
l)ut  a  little  more  slender  than  the  ventral  part.  The  chlo- 
rophyll contents  of  the  cells  make  the  archegonium  less 
transparent,  hence  for  the  most  part  the  ovum  and  the  ca- 
nal cells  of  the  neck  will  be  visible  only  after  the  sec- 
tion has  been  treated  with  potash.  In  the  axils  of  the 
perichaetal  leaves  are  many  short  paraphyses.  They  con- 
sist of  a  series  of  short  cells  somewhat  smaller  towards 
the  top,  the  lower  ones  often  being  brown. 

We  will  now  undertake  the  study  of  the  sporogonium 
of  this  plant,  Mniinn  Jwrnum.  It  is  the  so-called  "fruit  " 
of  the  moss.  It  consists  of  the  capsule  and  the  pedicel  or 
style.  The  bottom  of  the  latter  is  embedded  in  the  tissue 
of  the  mother  plant.  The  "  hood  "  (calypti  a)  which  arises 
from  the  enlarged  archegonium,  and  which  covers  the 
young  capsule,  will  be  cast  off"  very  early  and  will  there- 
fore be  diflicult  to  find.  It  is  composed  of  one  and  in 
part  also  of  two  layers  of  elongated  cells  and  is  split  on 
one  side  quite  up  to  its  slender  apex.  The  apex  ends 
in  a  brown  point  which  corresponds  to  the  neck  of  the 
archegonium.  At  the  bottom  where  it  was  broken  by 
the  growing  sporogonium  it  appears  as  if  cut  ofi".  The 
top  of  the  capsule  from  which  the  calyptra  has  been  re- 
moved is  covered  with  a  lid  which  is  provided  with  a  short 
beak.     It  may  easily  be  removed  by  means  of  a  needle 


SPOROGONIUM    OF   MOSSES.  273 

and  then  the  edge  of  the  urn-like  capsule  with  its  teeth 
come  into  view.  The  teeth  constitute  the  peristome.  The 
upper  part  of  the  style,  which  is  transformed  into  the  cap- 
sule, is  called  the  apophysis.  In  the  present  case  the  lat- 
ter is  separated  from  the  capsule  by  a  slight  contraction 
and  is  distinguished  by  its  brown  color.  In  some  of  the 
foliaceous  mosses  the  apophysis  is  much  stouter  than  the 
capsule,  as  in  the  jSplachnacece.  To  study  the  structure 
of  the  peristome,  w^e  should  cut  a  transection  of  the  cap- 
sule immediately  under  the  edge  of  the  urn,  and  transfer 
it  to  the  slide  with  the  teeth  upward.  Adjust  the  mirror 
and  view  the  object  with  reflected  light  using  a  low  mag- 
nification. We  observe  that  the  teeth  are  inserted  on  the 
inner  edge  of  the  urn,  are  pointed,  wedge-shaped  and 
striated  across.  If  we  breathe  lightly  upon  the  object 
during  the  observation  we  find  that  the  teeth  l)end  to- 
gether inward.  They  are  hygroscopic  and  bending  in- 
ward in  moist  weather  close  up  the  open  capsule,  while 
in  dry  weather  they  bend  back  outward  and  again  open  the 
capsule.  There  are  sixteen  teeth  upon  the  edge  of  the 
urn.  Put  such  a  section,  which  has  been  cut  open  on  one 
side,  in  a  drop  of  water,  laying  it  flat  down,  and  place  a 
cover-olass  over  it ;  examininar  it  with  transmitted  liaht, 
first  from  its  outside,  we  see  that  there  is  a  double  layer 
of  cells,  about  the  edge  of  the  urn,  which  is  composed  of 
inclined,  thickened,  papillaceous,  chlorophyll-bearing  cells 
with  colorless  walls,  browned  only  at  the  base,  where  they 
are  easily  detached  all  tooether  from  the  brown  edse  of 
the  urn.  These  cells  form  the  so-called  "rino;"  at  the  edoe 
of  the  capsule  and  mark  the  place  where  the  cover  sepa- 
rates. By  turning  the  inside  of  the  preparation  upwards, 
we  see  that  the  cross-markings  on  the  teeth  are  caused 
by  projecting  ledges  on  the  inside.  Inside  of  these  teeth 
are  the  so-called  "cilia."     Consequently,  this  plant  has  a 

18 


274  SPOROGONIUM   OP   MOSSES. 

double  mouth-piece,  while  the  Bryacem  possess  but  one. 
The  teeth  and  the  cilia  are  flat  lamellae  which  appear  to 
be  divided  off"  below  into  compartments,  and  to  be  cross- 
striped  above  with  low  projecting  ledges  on  the  inner 
surface.  Below,  it  is  united  into  a  continuous  membrane 
which  is  arched  a  little  between  each  two  teeth  of  the 
outer  mouth  parts.  Each  two  cilia  stand  between  two 
teeth  and  present  themselves  obliquely  from  the  edge. 
Their  edges  are  beset  with  serrate  projections,  the  outer 
along  their  whole  height,  the  inner  only  on  their  upper 
parts.  The  transverse  ledges  or  projections  of  the  free 
parts  of  the  cilia  end  in  these  teeth.  By  means  of  these 
teeth  the  ed<i'es  of  the  cilia  are  united  tos^ether  in  their 
upper  part  and  finally  form  a  long  slender  point.  Al- 
ternating with  these  pairs  of  hairs  are^  from  three  to  five 
very  slender  ones  which  stand  opposite  the  outer  teeth. 

A  delicate  transection  made  somewhat  deeper  through  the 
capsule  shows  within,  the  so-called  column  formed  of  large- 
celled  tissue.  About  this  column  lie  the  spore-filled  spaces. 
The  inner  walls  of  these  are  formed  by  the  column  itself, 
the  outer  by  a  double  layer  of  tissue  containing  chloro- 
phyll, which  is  separated  from  the  walls  of  the  capsule  by 
a  loose  tissue  of  cells  also  containing  chloroph^dl.  The 
walls  of  the  capsule  consist  of  two  to  three  layers  of  cells 
and  are  covered  by  a  distinctly  marked  epidermis,  the  cell- 
walls  of  which  are  much  thickened  on  the  outside.  The 
spores  contain  chlorophyll  grains,  their  walls  are  brownish, 
beset  with  warty  protuberances,  and  in  favorable  cases 
a  three-sided  pyramidal  outline  is  presented,  which  is 
caused  by  the  mother-cell  dividing  into  four  spores,  and 
these  flattened  pyramidal  surfaces  are  where  the  spores 
come  in  contact  and  press  upon  each  other  in  the  mother 
cell.  ; 

An  exact  median  longitudinal  section,  made  throuoh  a 


SPOROGONIUM    OF   MOSSES.  275 

green  full  grown  capsule,  on  which  the  cover  still  remains, 
will  show  that  the  latter  consists  of  a  layer  of  brown 
much  thickened  cells  without,  and  several  layers  of  thin- 
walled  cells  within.  On  the  boundary  between  cover  and 
capsule  lies  the  double  layer  of  obliquely-placed  chloro- 
phyll-containing cells,  already  known  to  us,  on  which  the 
separation  of  the  lid  from  the  capsule  depends.  The  next 
cells  of  the  capsule  walls  below  are  very  short.  Adjoining 
these  small  cells  on  the  inside  are  thickened  brown-colored 
cells,  which  form  an  inwardly  projecting  ledge  on  which 
are  set  the  outer  teeth  of  the  capsule.  About  one  cell 
thickness  beyond  arise  the  cilia.  The  history  of  the  de- 
velopment of  these  teeth  and  cilia  shows  them  to  have 
been  produced  by  local  thickening  of  the  opposite  walls 
of  one  and  the  same  cell  layer  on  the  inside  of  the  cover. 
The  teeth  arise  from  definite  portions  of  the  outer  wall 
which  are  connected  together  in  an  ascending  direction, 
whose  transverse  ledges  correspond  to  inner  adjoining 
transverse-walls  on  which  the  thickening  has  continued  a 
certain  distance  beyond.  The  cilia  arise  from  the  thickened 
parts  of  the  inner  walls  of  this  cell  layer  and  bear  low 
ledges  on"  the  places  nearest  the  attachment  of  the  inner 
division  walls. 

In  our  median  longitudinal  section  the  cover  is  hollow. 
After  the  completion  of  the  teeth  and  cilia,  the  inner  tissue 
shrivels  up  and  separates  from  the  inner  surface  of  the 
cilia  which  reach  to  the  apex  of  the  cover.  This  tissue 
forms  a  cone-shaped,  projecting  knob  on  the  top  of  the 
column.  The  latter  is  visible  alono-  its  whole  length.  We 
see  also  the  spore-sac,  its  outer  wall,  the  loose  tissue 
which  lies  between  it  and  the  wall  of  the  capsule,  finally 
the  latter  also.  The  spore-sac  is  closed,  while  the  cover 
remains  intact  by  a  thin  layer  of  tissue,  which  is  after- 


276  SPOROGONIUM   OF   MOSSES. 

wards  ruptured.  At  the  bottom  of  the  capsule,  under  the 
spore-sac,  a  ring-shaped  cavity  is  formed.  The  apophysis 
is  provided  with  stomata,  ahnost  every  median  longitudi- 
nal section  touching  one  of  them.  They  lie  under  the 
epidermis.  A  canal  leads  down  and  a  breathing  cavity  is 
at  the  end  of  it.  They  are  surrounded  by  chlorophyll-con- 
taining tissue,  whose  intercellular  spaces  communicate  with 
the  ring-cavity  at  the  base  of  the  spore-sac,  and  also  with 
the  air  cavities  in  all  the  chlorophyll-containing  tissue 
which  lies  between  the  spore-sac  and  the  wall  of  the  cap- 
sule. All  the  stomata  are  here  viewed  longitudinal!}'  and 
agree  in  form  with  those  of  the  vascular  cryptogams  and 
phanerogams.  The  apophj'sis,  and  in  other  cases  the  wall 
of  the  capsule  also,  furnish  the  only  places  in  the  mosses 
where  genuine  stomata  answering  to  those  of  the  higher 
plants  may  be  found. 

To  complete  our  view  of  the  structure  of  the  vessel  of 
the  moss-fruit,  we  will  make  a  superficial  section  of  the 
capsule  and  the  apophysis.  We  demonstrate  the  want  of 
stomata  in  the  surface  of  the  capsule.  Between  the  brown- 
walled  cells  of  the  apophysis  we  see  the  canals  which  lead 
down  to  the  stomata.  Turning  the  section  over  and  exam- 
ining it  from  the  inside,  we  shall  find,  in  favorable  cases, 
the  guard-cells  of  the  stomata  as  in  the  higher  plants. 
We  shall  also  observe  that  the  green  cells  lying  between 
the  spore-sac  and  the  walls  of  the  capsule  are  connected 
together  in  the  direction  of  their  length,  that  they  are 
branched,  and  altogether  appear  quite  like  filaments  of  al- 
gge.  A  transection  through  the  apophysis  mostly  touches 
those  stomata  whose  two  guard-cells  are  easily  seen .  The 
epidermis  proper  ceases,  the  surface  being  occupied  by 
two  or  three  layers  of  yellow-  to  red-brown,  much  thick- 
ened cells,  whose  inner  cavity  gradually  increases  in  size 


SPOROGONIÜM    OF    MOSSES.  277 

towards  the  interior  of  the  stem.  In  the  inside  of  the 
stem  a  conducting  bundle  is  differentiated.  A  median 
longitudinal  section  in  the  neighborhood  of  the  apophysis 
shows  that  these  relations  when  began  in  the  stem  very 
gradually  develop  themselves. 

Notes. 

(1)  Goebel,  die  Muscineen  in  Schenk's  Handbuch  der  Botanik,  Bd.  ii, 
p.  338. 

(2)  See  A.  Zimmermann,  Ueber  die  Einwirkung  des  Lichtes  auf  den 
Marchantienthallua,  Arb.  aus  d.  bot.  Inst,  in  Würzburg,  Bd.  ii,  p. 
665. 

(3)  Leitgeb,  Untersuchungen  über  die  Lebermoose,  vi  Heft,  1881, 
pp.  20,  117;  Goebel,  l.  c. ;  Strasburger,  Jahrb.  f.  wiss.  Bot.  vii,  p. 
409,  und  Befruchtung  und  Zelltheiluug,  1877,  p.  12. 


LESSON  XXVI. 
The    Reproduction    of    the    Vascular  Cryptogams. 

With  rare  exceptions  the  sporangia  of  the  ferns  are 
found  oil  the  under  side  of  the  leaf,  mostly  forming 
groups,  called  sori.  Often  the  whole  sorus  is  covered 
over  by  a  growth  from  the  leaf  called  the  indusium.  The 
indusium  is  yery  differently  developed  in  different  cases. 
Sometimes  the  edge  of  the  leaf  folds  over  the  sorus  form- 
ing what  we  call  a  false  indusium. 

Take  for  investigation  the  Scolopendrium  vulgai^e.  A 
prominent  mid-rib  runs  through  t^e  leaf  from  which 
branch  out  laterally  smaller  nerves  a  little  inclined  for- 
v^^ards.  The  sori  are  developed  on  the  upper  half  of  the 
fertile  fronds,  lying  in  the  same  general  direction  as  the 
lateral  nerves.  From  without  they  appear  to  be  covered 
more  or  less  perfectly  with  two  lip-like  indusia  which  at 
first  overlap  each  other  and  afterwards  gape  open.  Make 
a  transection  of  a  piece  of  the  fertile  frond  choosing  one 
whose  sori  have  become  brown,  but  whose  indusia  have  not 
yet  begun  to  gape.  Cut  out  a  piece  of  the  frond  with  the 
shears,  parallel  to  the  sori,  and  make  a  delicate  section 
by  means  of  elder-pith.  The  transection.  Fig.  94,  A^ 
shows  us  the  epidermis  of  the  upper  and  under  side,  and 
the  sponge-parenchyma  which  closely  joins  the  former. 
The  apparently  simple  sorus-stripe  is  found  to  be  double, 
the  parts  inclined  tow  ards  each  other  right  and  left,  and 
each  close  upon  a  vascular  bundle.  The  surface  of  the 
leaf  is  here  deeply  channelled,  with  a  projecting  edge  be- 
tween the  two  sori.  The  epidermis  at  the  bottom  of  the 
channel,  on  which   the  sporangia  are   growing,  lies  im- 

(278) 


REPRODUCTION   OF   FERNS. 


279 


mediately  iipoii  the  sheath  of  the  vascular  bundle.  The 
epidermis  of  the  under  surface  of  the  leaf  and  that  of  the 
canal  unite  to  form  the,  indusia,  ^,  i.  These  begin  in  a 
double  layer  of  cells,  but  soon  pass  into  a  single  layer 
which  has  the  structure  of  the  neighboring  epidermis,  ex- 


FiG.  94.  Scolopendrium  vulgare.  A,  transection  through  the  fertile  leaf;  i,  in- 
dusiiun;  sg,  sporansrium ;  B~E,  sporangia:  ß  and  E,  seen  from  the  side,  Ü,  from 
the  back  and  C,  from  the  front;  F,  a  spore.    A  X  50;  B-EX  145;  i^X  540. 

cept  in  lacking  stomata  and  chlorophyll  graius,  although 
colorless  chromatophores  are  found  in  it.  At  the  bottom 
of  the  channel  we  see  the  sporangia,  Sff,  in  different  stages 
of  development.  Each  arises  from  an  epidermal  cell. 
B}^  the  use  of  a  low  magnification.  Fig.  94,  A,  we  distin- 


280  REPRODUCTION   OF   FERNS. 

guish  in  each  sporangium,  a  style,  a  capsule,  and  iu  the 
older  ones  a  yellowish-brown  riug  on  the  capsule.  With 
a  higher  magnification  we  find  that  the  style  passes  from  a 
single  into  a  double  series  of  cells  and  the  walls  of  the 
capsule  consist  of  a  single  layer  of  cells.  Fig.  94,  B.  As 
the  different  views  of  the  wall  of  the  capsule  show,  B-E, 
the  rinor  is  formed  of  a  series  of  cells  in  the  wall  which 
project  outwardly,  beginning  at  the  style,  running  over 
the  top  of  the  capsule  to  the  opposite  side  where  they 
broaden  and  flatten  and  finrdly  end  without  reaching  the 
style  again.  The  inner  and  transverse  walls  of  the  ring- 
cells  are  much  thickened  and  browned,  the  thickening  on 
the  transverse  walls  diminishing  outwardly.  The  sporan- 
gium opens  between  the  broad  cells  iu  which  the  ring  ends. 
Fig.  94,  GE,  half  of  the  broad  cells  coming  on  one  side 
of  the  transverse  cleft  and  the  others  on  the  other  side. 
The  origin  of  the  springing  action  of  the  sporangium  lies 
in  the  ring  which  by  drying  produces  an  outward  tension. 
The  brown  walls  of  the  ripe  spores  show  a  beautiful  struct- 
ure, Fig.  94,  F.  The  outer  surface  is  covered  with  cox- 
comb-like projecting  ledges  united  together  in  a  netlike 
form. 

In  Aspidium  felix-mas  we  find  the  heart-kidney-shaped 
indusium  which  in  age  is  lead-colored  and  at  last  becomes 
brown  and  somewhat  shrunken  so  as  not  perfectly  to  cover 
the  dark  brown  sorus.  The  sporangia  have  the  same  struct- 
ure as  in  the  Scolopendrimn.  On  the  style  of  some,  a 
short  glandular  hair  is  found.  The  sporangia  grow  from 
a  cushion-like  elevation,  the  placenta,  which  lies  over  a 
vascular  bundle.  On  the  latter  are  placed  reticulated 
thickened  tracheids  which  extend  into  the  placenta.  At 
its  apex  the  placenta  bears  the  indusium,  inserted  with 
a  pedicel-forming  sinus.  By  making  a  preparation  of 
a ^'ipe  but  still  closed  sporangium  in  water  and  adding  a 


SPORANGIUM   OF   THE   FERN.  281 

dehydrating  fluid  like  glycerine  ut  tlie  edge  of  the  cover- 
glass,  the  sporangium  Avill  gradually  open  before  our  eyes, 
the  ring  becoming  strongly  concave,  after  which  a  backward 
movement  takes  place  by  which  the  sporangium  is  more 
or  less  perfectly  closed  again.  The  whole  ma}'  be  repeated 
with  diminished  force  several  times.  The  sporangium  of 
the  Scolopendrium  shows  the  closing  movement  much  less 
perfectly.  It  will  be  of  interest  to  examine  a  naked  sorus 
of  the  Polypodium  vulgare.  The  sori  of  this  genus  are 
entirely  without  indusia  and  lie  each  upon  avascular  bun- 
dle. The  placenta  rises  scarcely  above  the  surface  of  the 
leaf.  The  sporangia  are  of  the  same  type  as  those  of  the 
other  species. 

For  a  study  of  the  sexual  reproductive  organs  of  the 
vascular  cryptogams,  and  of  the  process  of  reproduction, 
we  will  also  select  a  fern.  The  prothallium,  the  first  sex- 
ually differentiated  generation  of  the  fern,  is  easily  ob- 
tained, either  by  sowing  the  spores,  or  collecting  the 
already  grown  prothallium.  For  the  latter  purpose  the 
Polypodiacece,  everywhere  occurring  and  rich  in  species, 
will  serve  us  best.  For  sowing,  the  spores  of  the  (7er- 
atopfei'is  thcdictroides,  cultivated  in  every  botanic  garden, 
may  be  chosen.  Like  that  of  most  other  Pohjpoddacecßy 
the  prothallium  of  Poli/podium  vulgare  has  the  form  of 
small,  heart-shaped,  living-green  leaflets  lying  on  the  sub- 
stratum. Seize  a  prothallium  of  medium  size,  at  the  point 
where  it  is  attached  to  the  substratum  and  remove  it 
from  its  fastening.  Wash  off*  all  the  adherino^  soil  in 
water  and  lay  it  on  a  slide  in  a  drop  of  water,  with  the 
ventral  side  up,  and  examine  under  a  cover-glass.  The 
heart-shaped  prothallium  consists  of  numerous  polygonal 
chlorophyll-bearing  cells.  In  the  anterior  sinus  lies  the 
small-celled  meristem  of  the  vegetative  point.  The  pro- 
thallium has  several  layers  of  cells  only  in  the  middle, 
the  so-called  tissue-cushion.    It  runs  out  on  the  sides  to  a 


-282 


PROTHALLIUM    OF   FERNS. 


single  layer  of  cells  and  gradually  flattens  itself  out  to- 
wards the  base  of  the  prothallium. 

The  root-hairs  or  rhizoids  spring  from  the  posterior  part 
of  the  frond,  being  found  mostly  in  the  middle.  They 
are  long,  single-celled  tubes,  soon  becoming  brown.  On 
the  under  edge  of  the  prothallium  single  cells  develop  into 
short,  almost  always  single-celled  papilhie,  which,  like  the 
rhizoids,  are  set  off  at  their  base  by  division  walls.  If 
we  have  a  young  prothallium  these  are  male,  if  an  old 
one,  they  are  exclusively  female  reproductive  organs.  Be- 
tween both  stand  those  which 
unite  the  two  sexes.  The  male 
organs,  the  antheridia,  are  found 
in  the  posterior  part  of  the  pro- 
thalliun,  among  the  root-hairs 
and  beyond  them  on  each  side. 
They  grow  at  the  apex.  They 
appear  as  spherical  arched  forms, 
Fig.  95,  A,  which  in  the  ripe 
state  contain  within  small  glob- 
ular cells  in  great  numbers.  Be- 
ripe    antheridia    are 

Ji  X  240.    C,  spermatozoid  in  mo-     ",  ,  .  .•     -i  i.i      • 

tion;  £>,  one  fixed  with  iodine  thosc  already  emptied,  as  their 
solution,  c and -DX  540.  browu  iuiier  walls,  and  a  star- 

shaped  hole  in  the  top  clearly  show.  We  get  a  com- 
plete view  of  the  structure  of  the  antheridium  only  when 
we  examine  it  in  profile.  This  view  may  be  got  by  bend- 
ing the  prothallium  over  a  needle.  This  view  is  seen  in 
Fig.  95,  A,  by  which  it  is  observed  that  the  antheridium 
sets  in  the  middle  of  a  low  arched  prothallium  cell,  2^, 
from  which  it  is  separated  by  a  division  wall.  The  wall 
of  the  antheridium  consists  in  almost  every  case  of  two 
stories  of  lateral  cells,  1  and  2,  and  a  cover-cell,  3.  The 
lower  has  a  wider  cell  cavity  than  either  of  the  upper  ones. 
The    side  view  of  an  empty   antheridium,  Fig.    95,  B, 


Fig.  95.  Poll/podium  vulgare.  A, 
a  ripe  and  B,  an  empty  antherid- 
ium; p,  protliallium  cell;  1  and 
2,  ring  ceils;  3,  cover  cell.  A  and     yond    the 


ANTHERIDIUM    OF   FERNS.  283 

shows  the  lateral  cells  much  swollen  and  veiy  prominent, 
the  inner  cavity  of  the  antheiidium  consequently  much 
diminished  and  the  cover-cell  pressed  flat  and  broken 
through.  If,  now,  we  return  to  the  superficial  view  of  the 
prothallium  and  examine  the  empty  antheridium  from 
above,  we  shall  see  that  the  side  cells  have  no  division 
walls,  and  we  perceive  that  they  are  really  ring-cells. 
The  whole  wall  of  the  antheridium  therefore  consists  of 
two  superimposed  ring-cells  a;id  the  cover-cell.  Cells  of 
this  kind  are  very  rare  elsewhere,  but  constantly  reappear 
in  the  antheridia  of  the  Pohjpodiacecß.  The  only  devia- 
tion from  this  form  of  antheridium  among  the  Poli/podia- 
cece  is  in  the  case  where  the  antheridium  is  l)orne  on  a 
pedicel  and  consists  of  but  one  ring-cell.  If  we  select  a 
prothallinm  which  has  not  been  wet  for  some  little  time,  we 
shall  not  have  to  wait  long  for  some  of  the  ripening  an- 
theridia to  discharge  their  contents.  The  mechanism  for 
the  discharge  of  the  antheridium  depends  upon  the  pres- 
sure which  the  ring-cells  exert  on  the  cell  contents,  as 
well  as  upon  the  swelling  substance  which  is  secreted 
among  the  peculiar  contents-cells  of  the  antheridium.  The 
cover-cell  will  finally  be  broken,  and  the  contents  of  the 
antheridium  will  be  pressed  out  and  the  ring-cells  will  in- 
crease in  size.  The  contents  consist  of  isolated,  spheri- 
cal cells,  the  spermatozoid  cells,  which  coming  out  into 
the  surrounding  water  remain  at  rest  for  a  little  while. 
As  may  be  seen  by  a  comparatively  low  magnification,  a 
little  filament  is  coiled  up  in  each  cell,  the  spermatozoid, 
and  a  central  collection  of  granules  may  also  be  recog- 
nized. The  walls  of  these  cells  after  a  few  hours  dis- 
solve in  the  surrounding  water  and  the  spermatozoids  are 
set  free.  This  takes  place  Avith  a  sudden  movement 
which  uncoils  the  spermatozoid.      One  spermatozoid  es- 


284  SPERMATOZOIÜS    OF   FERNS. 

capes  after  another.  We  follow  these  and  observe  that 
they  move  quite  rapidly  through  the  water  and  at  the  same 
time  rotate  about  their  axes.  After  al)out  twenty  or  thirty 
minutes  the  motion  begins  to  slacken  and  soon  ceases 
altogether.  During  this  last  stage  of  the  motion  of  the 
spermatozoid  its  form  is  not  difficult  to  recognize,  which 
may  be  done  all  the  more  easily  if  we  add  to  ihe  water- 
drop  containing  them  a  ten  per  cent  filtered  solution  of 
gum  arable  and  so  diminish  the  rapidity  of  their  motion 
(1).  The  spermatozoid,  Fig.  95,  (7,  is  formed  from  a 
band  rolled  into  the  form  of  a  corkscrew,  the  twist  being 
narrow  at  the  front  end  and  growing  wider  backwards. 
The  forward  end  bears  long,  fine  cilia.  Within  the  poste- 
rior twist  lie  fine  granules  and  often  an  inclosing  sac  may 
be  seen.  By  the  addition  of  a  little  potassic  iodide  of  io- 
dine solution  the  spermatozoids  are  very  beautifully  fixed. 
At  the  anterior  sinus  of  the  prothallium  we  shall  find 
the  female  reproductive  organs,  the  archegonia.  Next  to 
the  sinus  are  the  unripe  ones ;  beyond,  the  ripe  but  un- 
opened, and  beyond  them  still  the  opened  and  dead  ones 
brown  on  the  inside.  These  are  very  easily  distinguished 
from  the  male  organs.  They  rise  out  of  the  surface  of  the 
prothallium,  in  the  form  of  short  cylindrical  elevations  which 
bend  away  from  the  anterior  sinus.  These  free  parts  of  the 
archegonia  are  only  the  necks,  while  the  ventral  parts  are 
sunk  in  the  tissue  of  the  prothallium.  The  neck  is  com- 
posed of  a  wall  with  a  single  layer  of  cells  in  four  rows 
and  a  central  canal  whose  contents  in  the  ripe  archego- 
nium,  in  the  middle,  are  granular  and  in  the  periphery 
strongly  refractive.  This  inner  canal  widens  club-shaped 
above.  Below  it  passes  into  the  central  cell  of  the  arche- 
gonium  in  which  is  the  ovum.  The  latter  is  scarcely  dis- 
tinguishable.     If    we    do  not  wet   the   prothallium    for 


AECHEGONIUM    OF   FERNS.  285 

several  days  before  the  investigation,  we  shall  probably 
be  able  to  witness  the  opening  of  an  archegonium.  Take 
an  archegoninm,  the  contents  of  the  canal  of  which  are 
very  strongly  refractive.  The  opening  may  occur  in  a 
moment  or  we  may  have  to  wait  a  long  time  for  it.  The 
opening  of  the  neck  is  caused  by  the  pressure  of  the  re- 
fractive, expansive  substance  in  the  canal  on  the  walls  of 
the  neck.  The  four  cells  at  the  apex  of  the  neck  sud- 
denly separate  and  the  contents  of  the  canal  pour  out, 
distinfjuishinoj  themselves  as  a  colorless  mucilase  in  the  sur- 
rounding  water,  while  the  granular  contents  slowly  disor- 
ganize. The  emptying  of  the  archegonium  takes  place 
interruptedly,  first  from  the  neck  canal  and  then  from  the 
ventral  canal  cell  wdiich  lies  next  the  ovum. 

Under  especially  favorable  circumstances  one  may  see 
the  entrance  of  the  spermatozoids  into  the  archegonium. 
We  shall  increase  our  chances  of  this  it  we  use  an  old 
prothallium  having  archegonia  and  a  very  young  one  rich 
with  antheridia.  Spermatozoids  distributed  in  the  water 
swim  quietly  by  the  unopened  archegonia ;  but,  if  the 
archegonium  has  opened,  the  spermatozoids  for  a  measur- 
able distance  round  take  the  direction  of  the  open  mouth 
of  the  neck,  and  Avill  be  intercepted  by  the  mucilage  mass. 
Within  this  their  motion  lessens ;  still,  however,  keeping 
the  original  direction  and  lollow  down  the  neck  to  the  ovum 
in  which  they  are  absorbed.  As  has  been  lately  discov- 
ered, the  neck  of  the  archegonium  secretes  a  substance 
which  exerts  a  chemical  irritation  on  the  spermatozoid 
which  determines  the  direction  of  its  movement  (2). 
The  particular  irritating  medium  in  this  case  is  malic  acid, 
about  0.3  %  of  which  enters  into  the  mass  which  escapes 
from  the  neck  of  the  archegonium.  The  spermatozoids 
will  work  their  way  into  capillary  tubes  in  the  same  man- 
ner if  they  contain  a  0.01  %  to  0.1  %  solution  of  some 
base  united  with  malic  acid.     For  the  spermatozoids  of 


286 


ARCHEQONIUM   OF   FERNS. 


the  true  mosses,  cane  sugar  is  the  specific  irritating  medi- 
um, while  in  Marchantia  another  substance,  whose  nature 
is  not  yet  ascertained,  is  produced  by  the  archegonium. 

It  has  been  experimentally  demonstrated  that  (3)  a 
single  spermatozoid  is  sufficient  to  fertilize  the  ovum. 
Several,  indeed,  penetrate  into  the  archegonium,  usually, 
of  which  only  one  is  really  utilized.  These  processes, 
howev&r,  are  not  easily  followed  here  on  account  of  the 
lack  of  transparency  in  tke  tissue  of  the  prothallium. 
They  may  be  much  more  easily  seen  in  the  Ceratopteris. 
We  may,  however,  demonstrate  here  that  the  spermato- 


FiG.  96.    Poll/podium   vulgare.    ^,  uniipe  archegonium;  A",  neck  canal  cell; 
A'",  ventral  canal  cell;  o,  ovum;  B,  ripe  open  archegonium.  X  240. 

zoid-s  do  not  take  their  posterior  sacs  with  them  into  the 
archegonium,  but  leave  them  in  the  mucilage  in  front  of 
the  opening.  Sometimes  the  spermatozoids  are  so  numer- 
ous that  they  crowd  between  each  other  and  fill  up  the 
whole  neck-canal  with  a  filamentous  mass  and  form  besides 
a  tuft  about  the  opening. 

Finally,  we  will  examine  the  archegonium  in  section. 
Make  a  median  section  of  the  prothallium.  This  may  be 
facilitated  by  laying  several  of  them  together  after  first 
carefully  removing  every  adhering  grain  of  sand.  The 
archegonium  is,  as  we  see  in  Fig.  96,  A  and  B,  provided 
with  a  ventral  part  embedded  in  the  prothallium,  and  a 


SPORANGIA  OF  SELAGINELLA.  287 

curved  neck-portion.  The  neck-canal  cells,  W ,  and  the 
ventral-canal  cells,  K'\  are  now  distinguishable.  So  also 
the  ovum,  o,  with  its  nucleus.  The  ventral  part  of  the 
archegonium  is  inclosed  in  a  layer  of  flat  cells.  In  the 
ripe,  opened  archegonium,  B^  at  the  apex  of  the  ovum, 
there  is  often  seen  a  colorless  place,  the  embryo  sac,  where 
the  spermatozoid  is  received.  Other  sections  not  median 
v^Mll  give  us  a  sectional  view  of  the  antheridia. 

The  Selaginella  are  heterosporic  Lycojjodiaceoß.  They 
have  two  kinds  of  spores  and  sporangia,  which  we  will 
now  examine  in  order  to  complete  our  view  of  the  vascu- 
lar cryptogams.  The  Selaginella  are  also  called  ligulates 
because  their  leaves  are  provided  at  the  base  with  a  small 
tongue.  Take  Selaginella  Martensii  Sprg.,  a  greenhouse 
plant.  The  fertile  examples  are  easily  recognizable  by 
the  spikes  or  ears  borne  on  the  terminal  branches.  The 
vegetative  body  of  the  phmt  is  spread  out  in  a  plane, 
and  bears  four  rows  of  leaves  in  pairs  which  obliquely 
cross.  In  each  pair  the  upper  leaf  is  small,  the  lower 
considerably  larger.  The  two  series  of  upper  leaves  on 
the  back  side  press  against  the  stem  with  their  upper  side. 
The  two  series  of  under  leaves  on  the  ventral  side  are 
spread  out  flat  laterally,  with  their  upper  side  up.  The 
vegetative  bod}^  of  the  plant  is  therefore  bilateral  and 
dorsi-ventral,  that  is,  there  is  but  one  symmetrical  plane 
in  which  the  plant  is  laid  out  in  a  right  and  left  half,  and 
with  a  dorsal  and  ventral  surface.  The  fertile  terminal 
spikes  are  four-sided  and  provided  with  four  rows  of  leaf- 
flets  of  like  form  turned  upwards.  We  study  the  struct- 
ure of  the  spikes  in  this  wa}'.  Putting  it  under  the  sim- 
plex and  beginning  at  the  bottom  we  take  ofl'  one  leaf 
after  another  with  a  needle,  in  the  axil  of  each  of  which 
we  find  an  oval,  somewhat  flattened  sporangium.  We 
soon  see  that  many  of  the  sporangia  are  larger  than  the 


288  SPORANGIA  OF  SELAGINELLA. 

others  and  are  provided  with  a  projecting  knob.  If  Ave 
open  one  of  these  sporangia  with  a  needle,  four  large 
spores  which  perfectly  fill  the  sporangium,  and  whose  walls 
are  sometimes  arched,  make  their  appearance.  If  we  open 
one  of  the  smaller  sporangia  we  shall  find  it  filled  with 
numerous  small  spores.  The  larger  are  female  sporangia, 
macrosporangia ;  the  large  spores,  female  spores,  macro- 
spores.  The  smaller  are  male  elements  and  are  called 
microsporangia  and  microspores.  The  smaller  spores 
are  mostly  produced  in  fours,  and  have  three  flat  surfaces 
which  come  to  a  point  on  one  side  ;  on  the  other,  or  rounded 
side,  the  wall  is  beset  with  netlike  ridges.  We  meet  the 
same  relations  in  the  macrospores,  correspondingly  larger. 
The  walls  of  the  microspores  soon  become  dark  brown 
■while  those  of  the  macrospores  remain  much  clearer.  If 
we  observe  the  leaves  from  which  the  sporangia  have  been 
removed,  we  shall  see  the  ligula  as  tongue-shaped  mem- 
branes close  over  the  place  of  insertion  of  the  sporangia. 
A  further  removal  of  the  leaves  from  the  spike  shows  us 
that  the  macrosporangia  are  much  less  numerous  than  the 
microsporangia,  and  are  principal!}'  confined  to  its  lower 
part.  The  ripe  sporangium  opens  transversely  with  two 
lips. 

Herbarium  specimens  soaked  out  may  be  used  for  the 
study  of  the  vegetative  cone  and  the  sporangia.  Sections 
of  either  fresh  or  soaked  material  are  made  beautifully  trans- 
parent, by  the  use  of  potash  lye. 

Notes. 

(1)  See  Pfefler,  Uuters.  a.  cl.  bot.  Inst,  zu  Tübingen,  Bd.  i,  p.  370 

(2)  The  same  work,  p.  360. 

(3)  Strasburger,  Jahrb.  f.  wiss.  Bot.,  Bd.  yni,  p.  405. 


LESSON    XXVII. 
The  Reproduction  of  the  Gymnosperms. 

The  phanerogams  are  divided  into  two  large  groups, 
those  having  naked  seeds  and  those  having  covered  seeds, 
the  gymnosperms  and  the  angiosperms.  These  groups 
are  distinguished  by  differences  in  the  structure  of  the  flow- 
er, and  in  the  processes  of  fertilization  and  germ-building, 
which  we  will  consider  first  of  all  in  the  gymnosperms. 
We  shall  make  acquaintance  first  with  the  structure  of 
the  male  flower  (1)  of  the  fir  tree,  Pinus  sylvestris.  It 
ripens  the  pollen  towards  the  end  of  May.  Alcohol  ma- 
terial may,  however,  be  successfully  used,  but  on  account 
of  its  brittleness  it  should  be  soaked  out  a  few  days  be- 
fore using  in  a  mixture  of  like  parts  of  alcohol  and  glyc- 
erine. Material  thus  prepared  makes  far  better  sections 
than  when  fresh.  We  first  observe  that  the  male  flowers 
are  placed  in  large  numbers  on  the  under  parts  of  con- 
temporaneous or  growing  shoots.  They  are  arranged  in  a 
t\  oi'der,  and  correspond  in  position  to  that  of  the  two- 
needle  branches  which  adjoin  the  blossoms  in  an  inter- 
rupted series.  The  blossoms  like  the  leaf  bundles  occupy 
the  axils  of  the  secondary  leaves  or  scales.  Three  decus- 
sate pairs  of  scales  are  found  on  the  style  of  the  male 
flower.  The  lower  pair  is  laterally  placed  in  relation  to 
the  covering  scale  and  the  mother-shoot,  a  position  deter- 
mined by  the  existing  space  relations  involved,  a  position 
the  reverse  of  that  occupied  by  the  first  pair  of  leaves  of 
the  vegetative  buds  of  the  gymnosperms,  almost  without 
exception.  Next  to  the  scales  of  the  short  style  come  the 
stamens,  closely  compressed  and  mostly  arranged  in  ten 

19  Q289) 


290 


MALE   FLOWER   OF   FIR. 


regular  series.  The  axis  of  the  blossom  is  elongated  spin- 
dle shape.  A  single  stamen  removed  and  examined  with 
the  simplex  appears  round,  the  under  side  occupied  b}^  two 
pollen  sacs  longitudinally  inserted  and  meeting  along  a 
median  line ;  at  the  apex  runs  a  short  seam  which  extends 
upwards.  A  medum,  longitudinal  section  through  the 
blossom  shortly  before  the  flowering,  Fig.   97,  A,   and 


Fig.  97.  Pinus  pumilio  agreeing  with  Pinus  sylvestris.  D  from  P.  sylvestris. 
A,  longitmVmal  section  of  a  ripe  male  blossom.  X  W-  Lorigitudinal  section  of  a 
single  stamen.  X  20.  C,  transection  of  a  stamen.  X  27.  Z>,  a  ripe  pollen  grain.  X 
400. 

treated  to  potash  lye,  will  show  the  course  of  the  vascular 
bundles  in  the  axis  of  the  flower,  the  single  vascular  bun- 
dles with  which  each  stamen  is  provided  and  the  insertion 
of  the  pollen  sac  on  the  stamen. 

In  less  perfect  sections  thin  places  will  be  found  where 
the  structure  of  single  stamens  may  be  still  better  made 
out,  B.      By  making  a  tangential  section  of  the  blossom, 


POLLEN    OF    FIR.  291 

we  get  a  transection  of  single  stamens,  C,  which  we  will 
take  for  more  exact  study.     We  see  that  the  two  pollen  sacs 
come  togethei-  in  the  middle  and  when  ripe  are  separated 
by  a  flat  wall  of  compressed  cells  which  are  Anally  inter- 
calated by  one  or  more    layers    of  flat    cells    containing 
starch.     The   pollen  sacs  are  covered  with  the  epidermis 
on  their  free  surface,  on   the  inside  of  which  are  mostly 
compressed  cells.     In  the    niiddle  of  the  stamen,  in  the 
upper  and  under  part  of  the  wall  which  separates  the  two 
pollen  sacs,  runs  a  mesophyll  stripe.     The  upper  is  the 
larger  and  is  penetrated  by  a  v^ery  delicate  vascular  bün- 
dle.    On  the  lateral  edges  of  the  stamens  the  epidermis 
projects  in  the  form  of  minute  wings.     If  they  are  suffi- 
ciently large  they  contain  a  little  mesophyll.     On  the  un- 
der side  of  the  pollen  sacs  the  epidermal  cells  diminish  in 
size  from  both  sides,  and  at  the  point  of  least  development 
the  sac  opens.      These  pollen  sacs  very  closely  resemble 
the  sporangia  of  the  Lycopodiocece.     In  fact,  the  recent 
investigations  in  comparative  biology  have  led  to  the  con- 
clusion that  the  pollen  sac  of  the  phanerogams  and  the 
microsporangia  of  the  cryptogams  are  homologous  forms. 
If  we  now  examine  a  pollen  grain  produced  in  this  sac,  in 
as  fresh  a  condition  as  possible,  we  shall  see  that  it  has  a 
central  body  on  which  are  fixed  the  two  lateral  sacs,  D. 
In  the  ripe  blossom  these  will  appear  black  being  filled 
Avith  air.     Very  pretty  markings  are  seen  on  the  surface. 
The  inside  of  the  central  pollen    grain    proper   is  filled 
with  a  finely  granular  protoplasm  and  a  large  nucleus. 
Shortly  before    the  l)lossoming,  that   is  just  prior  to  the 
opening  of  the  pollen  sac,  the  pollen  grain  is  divided  hy 
a  wall,  shaped  like  an  hour-glass,  which  forms  a  lens-shaped 
cell  on  the  posterior  portion ;  that  is,  on  the  side  turned 
away  from  the  [)oint  where  the  wings  are  inserted.     This 
cell  is  best  seen  when  the  grain  lies  on  its  side  as  in  our 
figure.     A  cell  quite  like  this  is  diflerentiated  in  the  mi- 


292  STAMINATE    ORGAN    OF    JUNIPER. 

crospoie  of  the  heterosporic  PohjiJodiaceoe  before  the  be- 
ginning of  the  process  of  development  which  leads  to  the 
formation  of  the  sexual  product.  There  it  is  considered 
a  vegetative  cell  and  may  be  so  designated  here.  The 
wings  of  the  pollen  grain  are  produced  late  as^the  devel- 
opmental history  teaches,  by  the  elevation  of  the  cuticle 
between  which  and  the  inner  thickening  layer  of  the  wall 
a  watery  fluid  collects. 

"We  Avill  next  take  the  male  flower  of  Taxus  haccata. 
It  opens  in  March,  but  by  means  of  alcohol  material  one 
may  examine  it  at  any  time.  The  male  flower  is  found  in 
the  axils  of  the  leaves  of  last  year's  branches.  It  begins  with 
some  decussate  pairs  of  scales  which  pass  over  to  the  f 
arrangement.  The  scales  grow  constantly  larger  and  soon 
fall  into  a  quite  indefinite  arrangement  on  the  elongated 
axis  of  the  shield-shaped  stamen.  The  whole  bloom  as 
seen  with  a  magnifying  glass  resembles  not  a  little  the 
fertile  sporangia-bearing  leaves  of  the  Equisetum.  By 
removing  a  stamen  and  examining  with  the  simplex,  we 
shall  And  that  beneath  the  shield  are  inserted  from  five  to 
seven  pollen  sacs.  These  have  their  bas^  affixed  to  the 
under  side  of  the  shield,  and  their  inner  side  to  the  style. 
Laterally,  they  are  mainly  free  next  each  other,  and 
wholly  so  outwardly  and  at  their  apex.  Make  a  median 
and  also  a  tangential  longitudinal  section ;  the  former 
will  show  us  the  stamen  and  pollen  sac  in  longitudinal 
section  and  the  latter  in  transection.  The  pollen  sac  wi- 
dens outward,  the  section  showing  a  wedge-shaped  form. 
Both  sections  show  that  the  w^alls  of  the  ripe  pollen  sac 
are  reduced  to  the  epidermis  and  a  layer  of  compressed 
cells.  The  walls  of  the  epidermal  cells  are  provided 
with  thickened  ledges,  and  when  the  pollen  sac  is  separated 
from  the  style  the  epidermal  cells  show  a  considerable  re- 
duction in  size.  By  removing  a  pollen  sac  wall  from  the 
stamen,  with  a  needle,  we  shall  see  that  the  thickened 


PISTILLATE    ORGAN   OF   JUNIPER.  293 

ledges  on  the  inner  and  side  walls  of  the  epidermal  cells 
are  U-shaped.  This  thickening  occnrs  also  on  the  epi- 
dermal cells  of  the  outer  surface  of  the  shield.  The  pol- 
len sac  is  opened  by  the  walls  parting  from  the  style  and 
stretching.  The  pollen  grains  are  ellipsoidal  in  form  and 
beset  with  small  knobs.  Shortly  before  flowering,  the  end 
of  the  grain  is  diflerentiated  into  a  small  cell.  In  alcohol 
material  the  contents  of  the  pollen  grains  are  shrunken 
and  misery iceable  for  the  investigation. 

The  pollen  grains  of  Taxus  are  not  provided  with  the 
bladdery  inflations  of  the  walls  observed  in  the  Pinus,  nor 
do  they  occur  in  all  the  Abietince;  but,  on  the  contrary,  they 
recur  again  immediately  below  the  Taxus  in  the  Podocar- 
pus.  In  many  genera,  more  than  one  vegetative  cell  will 
be  differentiated  from  the  contents  of  the  pollen  grain 
whereby  projecting  cell  bodies  will  be  produced  within 
the  srain.  Among  the  Abietince  onlv  the  o-emis  Pinus 
has  simple  vegetative  cells. 

The  female  flowers  of  the  Taxus  baccata  (2)  are  found 
on  other  individuals,  since  the  plant  is  dioecious,  and  like 
the  male  flowers  in  the  axils  of  the  leaves  of  last  year's 
branches.  Fig.'  98,  A.  It  blossoms,  as  we  already  know, 
in  March,  but  alcohol  preserves  the  blossoms  very  well, 
and  such  specimens  serve  the  purpose  of  our  investigation 
perfectly  and  are  easily  managed  if  they  are  permitted  to 
lie  in  glycerine  and  alcohol  for  at  least  twenty-four  hours. 
The  blossoms  apparently  terminate  a  small  shoot,  but  are 
in  reality  not  terminal.  Not  seldom  we  find  two  flowers 
on  the  same  shoot.  Fig.  98,  at  *.  In  rare  cases  one  meets 
with  a  malformation  in  which  a  growing  foliage  shoot 
springs  out  of  the  side  of  the  blossom,  Fig.  98,  B.  AVith 
a  magnifying  glass  we  shall  perceive  that  the  floral  shoot 
begins  wifh  a  lateral  pair  of  scales  upon  which  follow  in  a 
spiral  order  other  scales  which  gradually  increase  in  size. 
The  blossom  itself  is  inclosed  in  three  decussate  pair  of 


204 


FEMALE    FLOWER    OF   JUNIPER. 


scales  from  which  only  its  protruding  apex  is  seen.  This 
apex  shows  a  piinctiform  opening,  the  micropyle.  We 
must  carefully  arrange  the  shoot  in  order  to  get  a  median 
longitudinal  section.  Tlie  section  should  be  made  through 
the  middle  of  the  pair  of  scales  which  stands  next  but 
one  beneath  the   blossom.     We  should  select  for  our  ex- 


no.  98.     Tnxiis  haccata.    A,  tj'pical  form  of  a  branch  with  female  flowers  at  the 
period  of  fevtillzalion  ;  at  *  are  two  ovules  on  the  same  primary  shoot,  natural  size. 

B,  a  k'af  with  a  floral  bud  in  its  axil,  the  primary  f-hoot  being  turned  aside.    X  2. 

C,  a  longitudinal  section  through  the  common  middle  of  a  primary  and  secondary 
shoot;  V,  vegetative  cone  of  the  primary  shoot;  a,  beginning  of  an  uxilhis;  e,  be- 
ginning of  an  embryo-sac;  n.  nucellus;  »,  integument;  n»,  micropyle.  X  18. 

amination  a  blossom  towards  the  end  of  April,  somewhat 
old  and  already  pollinized,  as  it  will  be  easier  to  cut  and 
will  in  many  respects  be  more  instructive.  If  the  section 
is  made  in  the  direction  indicated,  the  image  Avill  be  like 
that  represented  in  Fig.  98,   (7.     The  blossom  appears 


FEMALE  ORGAN  OF  JUNIPER.  295 

not  to  be  terminal  on  the  primary  shoot,  this  having  ter- 
minated its  (leveh)pment  by  the  formation  of  a  minute, 
secondary  shoot  in  the  axil  of  its  uppermost  scale,  which 
shoot  ends  in  the  flower  after  it  has  produced  three  decus- 
sate pairs  of  scales.  Laterally,  from  the  insertion  of  the 
secondary  shoot,  is  seen  the  vegetative  cone,  v,  of  the  pri- 
mary shoot,  pressed  to  one  side.  Here  and  there,  the 
next  scale  but  one  to  the  last  on  the  primary  shoot  forms 
a  secondary  shoot,  which  terminates  with  a  blossom  ;  and, 
in  rare  cases,  as  we  have  seen,  the  primary  shoot  forms 
foliage  leaves.  Fig.  98,  B.  The  pairs  of  scales  which 
precede  the  blossom  are  to  be  looked  upon  as  its  foliage 
envelope,  the  blossom  itself  being  reduced  to  an  ovule. 
We  see  one  of  them  in  the  form  which  terminates  the 
secondary  shoot.  We  distinguish  in  a  longitudinal  sec- 
tion the  following  parts  :  the  envelope,  i,  with  a  small 
opening  at  the  top  of  the  micropyle,  m,  and  within  this 
the  so-called  bud-nucleus,  the  nucellus  n,  in  the  bottom  of 
which  und^r  fiivorable  conditions,  by  treatment  with  pot- 
ash, we  may  recognize  a  large  cell,  the  beginning  of  the 
embryo  sac,  e  (3).  As  the  pollen  sac  corresponds  to  a 
microsporangium,  so  the  ovule  corresponds  to  a  macro- 
sporangium,  the  pollen  grain  to  a  microspore  and  the  em- 
bryo sac  to  a  macrospore. 

Biological  investigations  (4)  have  discovered  that  there 
are  important  resemblances  in  the  beginnings  of  these 
forms  ;  still  at  the  same  time  showing  that  a  progressive 
reduction  befalls  that  which  in  the  phanerogams  lead  to 
the  first  beginnings  of  the  macrospore.  On  the  other  hand, 
there  are  no  srounds  for  comparinof  the  integument  with 
the  indusium  of  the  vascular  cryptogams.  The  integument 
is  a  new  formation  appearing  on  the  macrosporangium  of 
the  phanerogams.  On  the  style  of  the  ovule  of  Taxus 
is  seen  a  small  tissue-mound,  a,  which  remains  stationary 


296  REPRODUCTION   IN   THE    FIR. 

for  a  long  time,  till  into  June,  but  afterwards  begins  to 
grow  and  in  the  fall  forms  the  brlMit  red  arilhis  which 
covers  the  ripened  seed.  On  the  already  pollinated 
bloom  which  we  have  taken  for  our  investigation,  we  may 
find,  lying  at  the  apex  of  the  nucellus,  a  pollen  grain, 
which  has  driven  a  short  tube  into  the  tissue  of  the  apex. 

It  is  the  large  cell  of  the  pollen  grain  which  is  grown 
out  into  this  tube,  while  the  small  vegetative  cell  is 
shrunken  up.  The  inner  covering  of  the  pollen  grain, 
the  intine,  forms  the  pollen  tube,  while  the  outer  integu- 
ment, the  warty  covering  of  the  ripe  pollen,  the  exine, 
will  be  stripped  off.  The  pollen  grain  lies  here  on  the  pa- 
pillose surface  of  the  apex,  while  i^i  other  species  of  Taxus 
and  its  near  relations,  the  apex  is  hollowed  out  (5)  to  re- 
ceive the  pollen  grain,  forming  the  so-called  pollen  cavity. 
If  we  would  learn  about  the  contrivance  by  which  the  pol- 
len grain  is  brought  into  the  ovule,  we  must  make  our 
observation  in  nature,  during  the  flowering-time  of  the 
plant  (6).  If  we  examine  a  female  jilant  at  the  time  the 
pollen  is  ripe  and  being  discharged  from  the  pollen  sac, 
we  shall  see  that  each  of  these  blooujs  secretes  a  little 
drop  of  fluid  from  its  micropyle.  The  pollen  grain,  borne 
by  the  wind,  falls  into  this  drop  of  fluid  and  is  sucked  in 
with  it. 

The  fir,  Pimis  sylvestris,  will  give  us  another  and  an 
extreme  example  of  the  structure  of  the  pistillate  blos- 
som in  the  conifers.  It  being  monoecious,  both  forms  of 
flowers  are  found  on  the  same  plant.  The  ovule  does 
not,  as  in  the  Taxus,  stand  alone,  but  a  cone  is  produced 
in  which  numerous  buds  are  united  together,  inserted  on 
scale-like  processes.  The  small  cones  occupy  the  tip  of 
the  present  year's  shoots,  singly  or  in  clusters.  They 
stand  in  the  axils  of  the  bracts,  like  the  lateral,  two-nee- 
dled branches  inserted  below  ;  but  their  position  above,  on 


CONE    OF   THE    FIR. 


297 


Sj-r 


the  shoot,  corresponds  to  that  of  the  branch  forming  long 
shoots.  The  small  cones  are  for  the  most  part  capable  of 
fertilization  by  the  end  of  May  and  are  noticeable  in  their 
relatively  smaller  size  by  their  brown-red  color.  They 
are  borne  upright  upon  a  stem,  the  stem  being  covered 
with  brown  scales.  Use  alcohol  material  which  has  been 
treated  with  glycerine  for  the  investigation.  Cut  away  the 
separate  parts  from  the  axis  of  the  cone  with  a  scalpel  and 
examine  them  under  the  simplex  separating  them  out  with 
a  needle  for  the  purpose. 

It  will  be  seen  that  standing  in 
the  axils  of  the  delicate,  reversed- 
oval,  enveloping   scales  with    a  'S^. 

fringed    edge,   Fig.    99,   &,    are 
similarly  shaped,  moie  thickened,     ,| 
smooth-edged  scales,/^  provided      |; 
on  the  inner  surface  with  a  me-      "^  -^j 
dian  projecting  carina,   c.     The  ,^^ 

latterare  the  seminiferous  scales.  ^'  j 

At  the  right  and    left  of  these 

1  i  4.1       1      ti  •      •  i     1         Fig.  99.    Pinus  sylvestris.  Sem- 

scales,  at  the  bottom,  is  inserted    ,^,,^^^^^^  ,^.,,^  ^,./^i,,^  ^,^  ,^.^ 

an    ovule  Avith    the    micropyle    of     ovules  .«  and  the  keel  c;  behind  is 

,     , .         J     ,    ,  ,  ,        ^        tlie   covering   scale,   6.     On  tlie 

each  directed  downward  and  out-      ovule   the  integnment  is  grown 

ward.  The  edge  of  the  integu-  out  into  two  processes,  m.  xv. 
ment  of  the  micropyle  is  elongated  into  two  right  and  left 
flaps,  m.  Bracts  and  seminiferous  scales  grow  together  at 
the  base  and  so  remain  attached  to  each  other  when  sepa- 
rated from  the  axis  of  the  cone.  The  cones  of  Abieti)ice 
and  other  conifers  will  be  considered  as  single  flowers  or 
as  a  mere  receptacle  for  flowers  according  to  the  signifi- 
cance which  one  attaches  to  the  seminiferous  scales.  They 
must  be  considered  either  as  flattened  metamorphosed  axil- 
lary shoots  partly  grown  to  a  bract,  or  as  a  placental 
growth  of  a  carpel  which  has  heretofore  been  known  as  an 


.«^ 

-:-'i^.. 


i^„—m 


298  SEMINIFEROUS    SCALES    OF    FIR. 

enveloping  scale.  In  the  first  case,  we  treat  each  as  a 
shoot  in  the  axil  of  the  bract,  bearing  two  ovules ;  in  the 
other  case,  we  consider  it  as  a  placenta  bearing  two  ovules, 
placed  on  the  upper  side  of  its  carpel.  In  the  first  instance, 
the  inflorescence  would  consist  of  a  cone  composed  of 
many  fertile,  axillary  shoots,  and  in  the  other  the  cone 
would  be  a  single  bloom  formed  of  numerous  carpels. 

The  remarkable  structure  of  the  seminiferous  scale  is 
explained  in  reference  to  the  act  of  fertilization  and  so 
can  be  followed  out  only  in  fresh  material  at  the  time  of 
pollination  (7).  As  soon  as  the  production  of  pollen 
begins  in  the  male  blossom,  one  will  notice  an  elongation 
of  the  axis  of  the  little  cones,  by  which  the  seminiferous 
scales  and  the  bracts  which  belong  to  them  are  separated 
a  little.  The  pollen  may  now  fall  upon  the  erect  seminif- 
erous scale,  slide  down,  and,  guided  by  the  carina,  come 
at  last  between  the  two  processes  of  the  integument. 
These  subsequently  roll  up  and  conduct  the  pollen  grains 
into  the  micropyle  and  to  the  embryo  sac.  After  being 
fully  pollinized  the  growing  seminiferous  scales  soon  glue 
their  edges  together  with  resin.  Neither  the  bracts,  nor 
the  carina  develop  farther,  the  latter  having  no  further 
function.  The  red  color  of  the  cone  passes  into  brown 
and  finally  to  green,  the  cone  gradually  taking  a  hanging 
position. 

We  shall  next  examine  another  variation  in  the  devel- 
opmentof  the  pollinized  ovule  of  the  conifers  (8) .  We  have 
already  learned  that  the  time  of  pollination  for  the  em- 
bryo sac  of  Taxus  is  in  the  first  beginning  of  it.  From 
this  followed  a  fiu'ther  development  of  the  ovule  so  that 
a  considerable  length  of  time  elapsed  between  the  polli- 
nation and  the  fructification  of  the  ovule.  In  Taxus  the 
fructification  takes  place  in  the  middle  of  June  of  the 
same  year ;   in  the  fir,  not  till   the  next  year,   thirteen 


OVULE    OF    RED-FIR.  299 

months  after  the  pollination.  In  the  pine,  the  two  acts 
are  separated  by  but  about  six  weeks.  We  shall  use  the 
fir  for  our  investigation.  It  would  lead  os  too  far  to  follow 
step  by  step  the  development  of  the  embryo  sac,  the  be- 
ginning of  the  prothallium  tissue,  the  endosperm,  and  the 
reproductive  organs  in  the  same,  the  increase  in  size  and 
consequent  diflerentiation  of  the  wdiole  ovule.  But  we 
will  take  it  at  the  point  when  the  ovum  is  fully  developed 
and  ripe  for  fertilization.  This  condition  is  reached  by  the 
common  or  red-fir,  Picea  vulgaris,  about  the  middle  of  June 
the  pollination  following  in  the  course  of  a  few  days. 
Alcohol  material  will  be  found  better  than  fresh  since  the 
ovum  will  be  tixed.  It  is  better  to  put  the  separate  scales 
rather  than  the  whole  cone  in  the  alcohol ;  and  the  material 
should,  as  heretofore  recommended,  be  previously  treated 
to  a  mixture  of  alcohol  and  glycerine,  equal  parts,  for  at 
least  four  and  twenty  hours  before  making  the  sections. 

We  should  first  take  a  general  view  of  the  whole  scale. 
It  is  an  inverted  oval,  on  the  iuner  surfiice  of  which  the 
beojinnings  of  the  two  seeds  are  seen  ;  also  the  outline  of 
the  wings,  which  afterwards  as  thin  lamella  of  tissue  will 
be  separated  from  the  inner  surface  of  the  seminiferous 
scale.  Beneath,  on  the  outer  surface  of  this  scale,  the  bract 
is  still  to  be  found,  now,  however,  quite  small.  We  may 
easily  remove  the  ovule  uninjured  from  the  seminiferous 
scale  w^ith  the  needle  in  order  to  make  a  section  of  it. 
Make  the  longitudinal  section  between  the  thumb  and  fin- 
ger. The  hardened  under  portion  of  the  ovule  will  not 
so  readily  lend  itself  to  section-making.  So  cut  away  the 
lower  half  with  the  shears  and  make  the  section  through 
the  soft  upper  part  containing  the  nucellus  and  the  embryo 
sac.  Stainino-  media  are  to  be  used  with  great  caution 
since  they   stain  the  whole  protoplasm  of  the   ovum  and 


500 


OVULE    OF    RED-FIR. 


nc 


easily  render  it  untr.insparent.  First  use  a  low  power  and 
as  we  are  looking  upon  a  median  section  cut  at  right  angles 
with  the  base  of  the  ovule  or  surface  of  attachment,  we 
shall  see  the  various  elements  as  represented  in  Fig.  100. 
The  integument,  ^,  forms  the  outer  envelope  of  the  ovule 

and  is  separated  from 
the  nucelhis  about  half 
Avay  up.  The  nucelhis 
bears  pollen  grains,  ^j, 
on  its  apex,  which  lie 
partly  within  and  partly 
without  the  tissue.  The 
tubes,  t,  from  these  pol- 
len grains  Avill  eventu- 
ally penetrate  the  upper 
part  of  the  nucellus  in 
order  to  reach  the  em- 
bryo sac,  e.  The  latter 
is  elliptical  in  outline 
and  tilled  with  endo- 
sperm, or  more  correct- 
ly prothallium  tissue. 
The  ventral  part  of  the 

Fig.  100.    Median  longitudinal  section  of  the  archegouia,    CI    (corpUS- 

fertilized  ovule  of  Pw-ea  rwi^'firis  Lk.  e,  embryo  ßnlj^")  mav  be  C'lsilv  rCC- 
sac  filled  with  endosperm;    a,  ventral  part  of  '.  '  '      J 

archegonium;  c,  neck  part;  »i,  nucleus  of  ovum;  Oguizcd  but  UOt  SO  easily 

nc,  nucellus  or  kernel  of  the  bud;  i>,  pollen  grain  .1  I-      .    .f      .     AVfl  * 

on  and  in  the  bud-nipple;  t,  pollen  tube  which  ^'^®  UeClv  part,  C.    VV  llnin 

penetrates  the  nucellus;  i,  integument;  s,  seed  eacll  archeo"Onium  is  an 
wings.  X9.                   .  *  T    .•  •   1 

ovum,  0,  (hstinguish- 
able  in  alcohol  material  by  its  yellow-brown  color,  in  the 
middle  of  which  is  a  nucleus,  n.  Finally,  the  attachment 
of  the  seed  wings,  s. 

If  we  make  a  section  through  a  fresh  specimen  we  shall 


OVULE    OF    RED-FIR.  301 

find  the  same  relations  again,  only  that  the  contents  of 
the  archegonium  will  often  be  discharged.  If  the  section 
touches  an  archegonium  without  opening  it,  the  ovum  will 
appear  as  a  yellowish,  foamy,  protoplasmic  mass  in  which 
the  nucleus  is  scarcely  distinguishable  or  at  best  has  the 
appearance  of  a  large  central  vacuole.  The  ovum  soon 
begins  to  sutfer  from  the  effects  of  the  surrounding  water. 
If  it  is  desirable  to  preserve  the  section  for  a  considerable 
time  it  is  recommended  to  use  diluted  white  of  an  egg  for 
an  examining  fluid,  and  to  make  this  still  more  durable  a 
little  camphor  may  be  added  to  it  (9).  In  such  prepara- 
tions, the  neck  part  of  the  archegonium  is  not  difficult  to 
see.  It  consists  of  from  two  to  four  stories  of  cells.  Un- 
der the  neck  part  is  a  small  cell  which  corresponds  to  the 
ventral  canal  cell  of  the  vascular  cryptogam,  the  ovum 
parting  to  form  it  shortly  before  ripening.  The  ventral 
part  of  the  archegonium  is  surrounded  by  a  layer  of  flat 
cells  rich  in  contents,  like  the  covering  which  we  saw 
about  the  ventral  part  of  the  fern.  In  order  to  know  the 
number  and  position  of  the  archegonia  we  must  make  a 
series  of  transections  through  the  upper  part  of  the  ovule. 
By  this  means  we  shall  see  that  from  three  to  five  arche- 
gonia are  arranged  in  a  circle  at  the  apex  of  the  embryo 
sac.  Sections  which  touch  the  apex  of  the  embryo  sac 
present  us  with  an  apical  view  of  the  neck  part  of  the  ar- 
chegonia which  is  a  rosette  of  from  six  to  eight  cells.  If 
our  material  has  been  collected  at  the  period  of  fertiliza- 
tion we  shall  eventually  find  pollen  tubes  which  have 
penetrated  to  the  ovum,  and  in  the  under  part  of  single 
ovules  we  shall  find  a  four-celled  rosette  which  may  be 
traced  out  into  prothallium  tissue  in  four  uninterrupted 
tubes.  The  four  terminal  cells  of  these  tubes  produce 
the  germ.  The  seeds  ripen  in  October.  It  separates  then 
easily  from  the  seminiferous  scale.     The  wings  continue 


302 


EMBRYO    OF    RED-FIR. 


Oil  the  inside  of  the  seed  bet^reen  them  and  the  seminif- 
erous scale,  the  seed   falling   off  later  from    the  Avmgs, 
leaving  a  corresponding  cavity  in  the  same.     Sections  in 
both  directions  will  show  that  the  cells  of  the  seminifer- 
ous scales  are  so  thickened  as  nearly  to  obliterate  their  cell 
cavity.     A  part  of  the  prothallium  tissue  is  filled  WMth 
reserve  material  which  has  been  preserved  as  seed  albu- 
men or  endosperm  in  the  seeds.     It  forms 
a  sac  inclosing  the  germ.     This  sac  is  open 
on  its  micropyle  end  and  here  the  root  end 
of  the  seed  joins  the  remainder  of  the  sup- 
planted nucellus.     The  germ  appears  like 
a  cylinder  which  grows  thicker  toward  the 
end  of  the  cotyledon,  and  in  consequence 
of  being  filled  w^ith  albumen  is  white  and 
untransparent  like  the  cotyledons.     Make 
a  lono-itudinal  section  between  the  fingers 
and   examine  it  in  carbolic  acid    diluted 
with  alcohol.    This  makes  the  image  very 
clear,  far  more  so  than  potash  lye  or  even 
chloral  hydrate  itself,  so  that  each  row  of 
cells  may  be  easily  followed.      We    see 
Fig.   101,  c,  that  the  cotyledons    do  not 
reach  quite  a  third  of  the   length  of  the 
germ  and  between  them  at  the  base  is  the 
vegetative  cone  of  the  stem.     The  little 
stem  or  cauliculus  which  will  be  designated 
the  hj'pocotyledon  or  h3'pocotyle<lonous  link,  It,  is  a  pos- 
terior continuation  without  distinct  limits  of  the  rootlet 
(radicle).     This  is  mainly  discernible  only  l)y  means  of  a 
vegetative  cone  which  shows  itself  distinctly  within  the 
body  of  the  germ  as  the  apex  of  the  plerome,  of  the  root 
pi,  while  the  cell  rows  of  the  rind  of  the  hypocotyledon 
continue  directly  into  the  parabolic  layers  of  the  root-cap, 


Fig.  101.  ricea  vul- 
garis. Longitudinal 
section  of  tlie  ripe 
germ;  c,  cotyledon^ 
ft,  hypocot.\ledonoiis 
member;  pi,  apex  of 
plerome;  cp,  root 
cap ;  cl,  middle  col- 
umn of  llie  same;  m^ 
pith;  o;»,  procambium 
ring  in  tlie  liypocoty- 
ledonous  member.  X 
10. 


EMBRYO    OF    RED-FIR.  303 

cp,  a  process  which  we  find  repeated  in  all  roots  of  the 
gymnosperms  wherever  Ave  can  see  the  cell  rows  of  the 
rind  of  the  root-body  pass  over  directly  into  the  cell  lay- 
ers of  the  root-cap.  See  TJnda,  p.  177.  The  root-cap  is 
penetrated  in  its  long  axis  by  a  pith-like  column,  cl,  of 
cells  arranged  in  straight  rows.  In  the  hypocotyledon,  the 
tissue  of  the  pith,  m,  already  begins  to  show  itself,  and 
about  this  the  elongated  cells  of  the  procambium  ring,  op, 
in  which  the  vascular  bundles  appear.  These  cells  may 
be  plainly  seen  in  a  median  section  of  the  cotyledon.  See 
the  figure.  Thus  we  see  that  the  essential  parts  of  the 
future  plant  already  appear  in  the  embryo. 

Notes. 

(1)  See  Strasburger,  Coniferen  u.  Gnetaceen.  p  120;  Eichler, 
Bliitheiidiagramme,  Bd.  i,  p.  58;     Goebel,  Gruiidziige,  p.  363. 

(2)  Strasburger,  Coniferen  u.  Gnetaceen,  p.  2. 

(3)  Strasburger,  Angiosp.  uud  Gymnosperm,  p.  109. 

(4)  Strasburger,  same  work,  p.  109;  Goebel,  Bot.  Ztg.,  1881,  sp. 
681. 

(5)  Strasburger,  Jenaische  Zeitschr.  f.  Naturw.,  Bd.  vi,  p.  251 ; 
Conif.,  u.  Gnet.,  p.  250. 

(6)  Same  worlj,  pp.  250  and  265. 

(7)  Strasburger,  last  work,  and  Vol.  quoted,  p.  251 ;  Conif.  u. 
Gnet.  p.  267. 

(8)  See  Strasburger,  Befr.  b.  d.  Conif. ;  Couif.  u.  Gnetaceen,  p. 
274;  Befr.  u.  Zellth.  a.  v.  O.  Angiosp.  u.  Gymnosp.,  p.  140;  Goroschau- 
kiu,  Ueber  die  Corpusculau.  d.  Befr.  bei  d.  Gymuosp.  russisch.,  1880. 

(9)  Strasburger,  Befr.  b.  d.  Conif.,  p,  8. 


LESSON  XXVITI. 
The  Andrceceum  in  the  Angiosperms. 

The  collective  male  organs  of  an  angiosperra  blos- 
som form  the  andrceceum.  The  pollen  vessel  or  pollen 
leaf  (stamen)  (I)  consists  of  a  filamentous  support,  the 
filament,  and  the  anther.  The  latter  is  formed  of  two 
parts  lying  side  by  side  lengthwise  and  separated  by  the 
upper  part  of  the  filament,  the  so-called  connective  tissue. 
This  tissue  it  is  best  to  reckon  as  a  part  of  the  anther. 
Two  pollen  sacs  are  commonly  embedded  in  the  tissue  of 
each  half  of  the  anther.  Each  of  these  sacs  or  compart- 
ments correspond  to  a  microsporangium.  For  a  study  of 
the  stamen  we  will  take  in  the  first  case  a  large  flowered 
lily,  as  for  example,  the  Hemerocallis  Julva  cultivated  ev- 
erywhere in  gardens.  The  yellow  filament  is  very  long 
and  becomes  slenderer  above  and  is  very  sharply  pointed 
at  the  place  of  insertion  in  the  anther.  The  anther  is 
brown  and  movable  on  the  filament.  The  connective  tis- 
sue may  be  traced  as  a  narrow  stripe  on  the  outer  surface 
of  the  anther  between  the  two  lobes.  The  ripe  pollen 
may  be  examined  on  the  slide.  It  has  the  form  of  a 
coffee  bean.  It  is  yellow  and  its  surface  is  covered  with 
reticulated  ledges.  If  a  little  water  be  introduced  from 
the  side  of  the  cover-glass,  the  crease  or  fold  in  the  pollen 
grain  will  soon  disappear  and  the  grain  on  that  side  swell 
out  till  it  takes  the  form  of  an  ellipsoid  flattened  a  little 
on  one  side.  The  membrane  of  the  before-infolded  part 
is  relatively  of  considerable  thickness,  colorless,  without 
markings  and  is  very  sharply  defined  against  the  brown- 

(304) 


DEVELOPMENT    OF    TOLLEN.  305 

ish-marked  membranous  part.  By  careful  focussiug  on  a 
favorably  placed  pollen  grain  we  learn  that  it  is  enclosed 
in  a  simple  membrane,  that  the  colorless  part  thins  out  at 
the  edges  and  passes  directly  into  the  colored  part.  Be- 
tween the  grains  in  the  preparation  and  also  adhering  to 
their  surfaces  are  orange-red  oil  drops  which  in  their  dry 
state  give  them  their  yellow  color.  The  contents  of  the 
pollen  grain  are  gray  and  finely  granular.  After  a  short 
time,  during  which  the  grain  gradually  enlarges,  it  bursts 
and  empties  its  contents  vermitormly  iu  the  surrounding 
water.  If  a  solution  of  sugar  of  the  proper  concentration 
be  used  the  pollen  grains  will  round  out  and  not  burst  and 
may  be  examined  unhindered.  Treating  with  concentrated 
sulphuric  acid,  the  smooth,  colorless  portion  of  the  Avail 
slowly  dissolves,  l)ut  the  striated  colored  portion  resists 
the  action  of  the  acid ;  it  is  cutinized.  The  cutinized 
membrane  of  pollen  grains  which  have  an  infolded  part 
protects  the  whole  grain  when  the  anther  is  open.  As 
may  be  seen  in  the  dry  grain  the  edges  of  the  cutinized 
portion  of  the  membrane  touch  each  other  along  the  whole 
length  of  the  fold  so  that  the  uncutinized  part  lies  wholly 
buried  under  it  in  the  fold.  It  comes  to  view  only  when 
the  pollen  grain  is  placed  on  the  stigma,  begins  to  swell 
and  puts  out  the  pollen  tube.  But  in  the  pollen  of  this 
plant  an  exine  and  inline  or  special  outer  and  iimer  part 
is  not  to  be  distinguished,  since  the  wall  is  nowhere 
double.  Its  cutinized  part  has  the  function  of  exine,  and 
the  uncutinized  of  intine  in  other  pollen  grains.  By  the 
action  of  sulphuric  acid  the  structure  of  the  cutinized 
membrane  is  very  distinctly  seen.  By  strong  magnifica- 
tion and  looking  at  it  from  above,  we  see  a  wandering  net- 
work with  delicate  wavy  walls.  In  some  of  the  meshes 
lies  a  blue  irregularly  shaped  body  which  is  the  oil  drop, 
before  yellow  but  now  colored  blue  by  the  acid.     The  cu- 

20 


306 


DEVELOPMENT    OF    ANTHER    OF    LILY. 


tiiiizetl  meml)rane  itself  is  colored  3ellow.  By  a  median 
focus  it  is  easy  to  see  a  compound  inner  wall-layer  on 
which  rest  the  outer  projecting  ledges.  The  ledges  them- 
selves are  swollen  on  their  outer  edges  so  that  in  optical 
transection  they  appear  club-shaped.  In  a  superticial 
view  we  find  that  the  ground  surface  is  covered  with  tine 
points  the  optical  transection  of  which  demonstrates  them 
to  be  in  reality  small  knobs  resting  on  the  inner  wall-layer. 


•  -cC»— ' 


V-v 


Fig.  102.  HemerocnHis  fulva.  A,  transection  of  an  almost  ripe  anther,  the  cut- 
ting having  opened  the  pollen  chambers;  p.  the  division  walls  between  the  com- 
partments; /,  vascular  bundle  in  tlie  connective;  a,  furrow  in  the  connective.  X 
14.  B,  cross-section  of  a  young  aniher.  X  '-S.  C,  part  of  the  last  preceding  sec- 
tion, the  tissue  over  a  chamber;  e,  epidermis;  f,  cells  which  afterwards  form  the 
fibrous  layer;  c,  a  cell  layer  which  will  disappear;  t,  tapestry  layer  which  after- 
wards dissolves;  pm,  pollen  mother-cells.  X  210.  Z»  and  £,  pollen  mother-cells 
which  have  undergone  self-division.  X  210. 

After  being  subject  to  the  action  of  the  acid  for  some 
hours  the  pieces  of  the  membrane  take  on  a  reddish-brown 
color  while  the  contents  of  the  pollen  grain  are  at  the  same 
time  tinged  a  rose-red,  a  behavior  which  protoplasm  often 
shows  towards  sulphuric  acid  (2).  In  25 (/o  chromic  acid 
the  uncutinized  membrane  and  the  contents  of  the  grain 
are  rapidly  dissolved,  the  cutinized  part  resisting  longest, 


ANTHER    OF    HEMEROCALLIS.  307 

the  network  of  ledges  alone  remaining  and  these  finally 
disappearing. 

We  will  now  make  a  transection  of  the  anther  which  we 
can  do  best  by  taking  a  two-thirds  grown  flower-bud  and 
cuttino-  thronoh  the  whole  of  it.  With  the  needle  remove 
the  sections  of  the  perigoneal  leaf  from  the  preparation. 
Notwithstanding  we  have  taken  so  young  a  flower,  we  shall 
find  all  the  compartments  of  the  anther  open.  The}^  open 
very  easily  and  the  pressure  of  the  knife  in  cutting  the 
section  has  done  it.  Figure  102,  Ä,  represents  the  sec- 
tion. The  walls  of  the  two  pollen  chambers  separate 
themselves  from  the  division  Avail  at  j)-  f^hey  unfold 
their  bent  forms.  The  two  halves  of  the  anther  are 
joined  together  by  a  slender  connective  containing  a  vas- 
cular bundle,/".  Examined  with  a  high  magnification,  we 
find  a  flat-celled  epidermis  on  the  outside,  filled  with  vio- 
let cell-sap.  These  epidermis  cells  are  l)owed  outward. 
On  the  edges  of  the  chamber  walls  the  height  of  these 
cells  rapidly  diminishes,  and  at  this  point  the  middle  di- 
vision wall  ruptures.  Stomata  are  distributed  over  the 
whole  surface  of  the  anthers,  a  small  breathing  cavity  lying 
under  each.  Next  succeeding  to  the  epidermis  on  the  walls 
of  the  com[)artments  is  a  single  layer  of  relatively  high  cells 
provided  with  ring-shaped  thickenings,  the  so-called  flbrous 
layer.  The  rings  of  these  cells  are  placed  perpendicular 
to  the  surface,  sometimes  pass  into  spirals  and  anasto- 
mose in  many  ways  with  each  other.  Towards  the  back 
of  the  anther  the  walls  of  the  compartments  become  grad- 
ually thicker,  the  layer  of  fibrous  cells  being  doubled. 
The  rest  of  the  body  of  the  anther  is  also  built  of  these 
fibrous  cells.  Only  those  cells  which  surround  the  vascu- 
lar bundle  in  the  connective  and  those  which  form  the  di- 
vision walls  between  the  chambers  of  the  anther,  jj,  are 
without  thickened  ledges. 


308  DEVELOPMENT   OF   POLLEN. 

We  must  take  a  two-thirds  grown  flower-bud  in  order 
to  get  a  good  superficial  section  of  the  anther.  This  Avill 
show  us  that  tlie  epidermal  cells  are  elongated  longitudin- 
ally over  the  compartments  while  those  of  the  fibrous 
layer  are  extended  perpendicular  to  the  surface.  But  on 
the  back  side  of  the  anther  the  fibrous  cells  are  more  nearly 
isodiametric.  Over  the  pollen  chambers  the  thickened 
ledges  of  the  fibrous  cells  are  much  weaker  on  the  outside 
of  the  walls,  often  scarcely  discernible,  while  the  lamellee 
of  the  thickened  ledges  on  the  inside  next  the  cell  space  are 
drawn  closer  together  than  those  of  the  outside  by  drying. 
This  causes  the  walls  of  the  compartments  to  spring  back 
when  they  are  ruptured.  Frequently,  in  the  angiosperms, 
as  in  Taxus,  the  thiclvenings  of  the  outer  walls  of  the  fibrous 
cells  in  the  walls  of  the  compartments  cease  altogether, 
so  that  the  fledges  become  U-shaped  or  basket-shaped, 
opening  outward.  It  is  clear  that  such  an  arrangement  fa- 
facilitates  the  rolling  back  of  the  tissue  forming  the  com- 
partment walls.  In  order  to  understand  the  relation  of 
the  filament  to  the  anther,  we  must  make  a  median  longi- 
gitudinal  section  of  the  upper  part  of  the  stamen  between 
the  two  halves  of  the  anther.  We  see  that  the  filament  is 
very  much  attenuated  at  the  point  where  it  is  inserted  in 
the  anther.  The  cells  which  surround  the  vascular  bundle 
may  be  traced  from  the  filament  into  the  connective.  They 
are  not  fibrous  cells.  In  order  to  o-et  a  section  of  the  an- 
ther  with  its  chambers  closed,  Fig.  102,  B,  we  must  take 
a  sufiiciently  immature  dower-bud  for  our  section. 

If  now  we  make  a  transection  of  a  floral  bud  only  6  or 
7  mm.  high  we  shall  find  in  the  walls  of  the  chambers  be- 
sides the  epidermis,  Fig.  102,  O,  e,  two  or  three  layers  of 
flat  cells,  f,  c,  and  one  of  radially  elongated  cells,  t.  The 
latter  inclose  the  whole  chamber,  the  interior  of  which  is 
filled  with  polygonal  pollen  mother-cells,  /JWi. 


DEVELOPMENT   OF   POLLEN.  309 

By  making  a  section  of  a  bud  1  cm.  long,  we  shall  find 
the  pollen  mother-cells  already  isolated  and  in  the  act  of 
self-division.  They  are  recognizable  by  their  thick,  white, 
strongly  refractive  walls,  their  contents  being  already  di- 
vided into  from  two  to  four  cells  which  lie  in  one  plane, 
Fig.  102,  Z>,  or  in  two  planes  at  right  angles,  Fig.  102,  E. 
These  become  subsequently  the  pollen  grains,  produced, 
like  the  spores,  by  fourfold  division  within  their  mother- 
cell.  The  walls  of  the  anther  are  lined  with  so-called 
"tapestry  cells",  t,  which  are  filled  with  yellow-brown  con- 
tents, and  arise  from  the  innermost  of  the  layers  lining  the 
chamber.  In  the  next  older  flower-bud  the  walls  of  the 
pollen  mother-cell  are  dissolved,  the  young  pollen  grains 
lie  free,  the  tapestry  cells  have  for  the  most  part  given  up 
their  independent  existence,  their  contents  being  pressed 
in  between  the  young  pollen  grains.  The  layer  of  flat 
cells, y,  lying  immediately  beneath  the  epidermis  is  much 
developed  and  forms  the  fibrous  layer,  while  the  next  in- 
ner layer  has  become  compressed  and  disorganized.  An 
older  bud  will  show  that  the  still  unused  tapestry  cells, 
especially  in  the  periphery  of  the  chamber,  have  an  intense 
yellow-brown  color,  and  oily  sparkling  appearance,  and 
thus  form  the  oily  substance  which  surrounds  and  adheres 
to  the  pollen-grains. 

All  species  of  lilies  behave  like  the  Hemerocallis,  but 
the  diflerentiation  of  the  anther  comes  later.  The  pollen 
mother-cells  begin  to  divide  in  Liliu7n  candidum,  L.  cro- 
ceum  and  other  species,  only  when  the  flower  bud  is  2  cm. 
high.  In  transections  of  fresh  buds  the  large  tapestry  cells 
will  be  very  noticeable  with  their  yellow-brown  contents. 
The  hypodermal  cells  as  well  as  all  others  which  are  after- 
wards to  be  provided  with  ledge-like  thickenings  are  filled 
with  starch  grains. 

Funkia  ovata  is  a  good  plant  for  study,  and  behaves  like 
Hemerocallis  and  Lilium;  so  also  Agcqxmthus  umbellatus. 


310 


POLLEN    OF    TRADESCANTIA. 


Tulipa  und  Hyacinthus  orientalis  are  likewise  good  ob- 
jects. In  Tulipa  the  filament  is  so  much  attenuated  under 
the  anther  that  the  latter  will  rotate.  In  the  hyacinth  the 
anther  almost  sits  on  the  perigone. 

Tradescantia  Virginica  is  less  easy  to  make  sections  of, 
but  we  will  examine  it  in  reference  to  the  pollen  grains. 
A  transection  of  a  bud  about  two-thirds  grown  shows  us 
the  tAvo  halves  of  the  antlier  separated  b}'^  a  rather 
thick  connective.  The  walls  of  the  compartments  are  al- 
ready reduced  to  two  layers  of  cells,  and  the  thickened 
ledges  are  already  developed  on  the  inner  one.  The  pol- 
len   grains  are    embedded    in  a   yellow-brown  substance 

with  whose  origin  from 
the  tapestry  cells  we  are 
already  acquainted.  The 
division  wall  between  the 
two  chambers  is  so  fully 
developed  and  is  so  thick 
that  there  is  scarcely  any 
depression  observable 
between  them.  At  the 
point  of  insertion  of 
the  chamber  wall  upon 
the  division  wall  the  fibrous  layer  suddenly  ceases,  and  at 
this  point  the  separation  takes  place  later  on.  An  exami- 
nation of  the  surface  of  the  chamljer  wall  shows  in  this  case 
a  longitudinal  diminution  of  the  epidermal  cells  and  a 
transverse  lessening  of  the  fibrous  layer  with  a  most  com- 
plete faikn-e  of  the  thickened  ledges  on  the  outside  walls 
of  the  cells. 

If  Ave  examine  with  the  maijnifier  the  stamen  of  a  bud 
just  ready  to  blossom  out,  we  shall  see  the  beautiful  yel- 
low anther  on  the  violet  colored  filament  beset  Avith  violet 
hairs.  The  dry  pollen  grains  are  noAV  folded  on  one 
side,  Fig.  103,  A.    In  Avater,  the  fold  is  smoothed  out 


Fig.  103.  Tradescantiavirginica.  A,  pollen 
grain  dry;  B,  in  water;  C,  j'oung  pollen  grain 
in  water  showing  the  vegetative  cell.  X  -^^0. 


STUDY    OF    POLLEN.  311 

and  the  grain  becomes  ellipsoidal  in  shape,  the  previously 
folded  side  being  more  convex  than  the  other.  The  mem- 
brane is  striated  with  meandering  lines.  The  folded  side 
shows  this  structure  also  and  is  distinguished  only  by  its 
brighter  color  and  its  somewhat  weaker  cutinization.  In 
the  finely  granular  contents  are  to  be  distinguished  two 
clear,  homogeneous  spots,  B.  They  are  the  two  nuclei,  of 
which  one  is  vermiform  and  the  other  ellipsoidal.  The 
rest  of  the  contents  of  the  pollen  grain  is  pretty  uniformly 
fine-grained.  The  pollen  grain  very  soon  begins  to  flatten, 
whereby  the  nuclei  together  with  the  rest  of  the  contents 
are  much  compressed.  Both  nuclei  may  be  seen  very  beau- 
tifully if  the  pollen  grain  be  crushed  in  a  drop  of  acetate 
of  methyl  green,  or  acetate  of  iodine  green.  The  ver- 
miform nucleus  is  deeply  stained  and  much  elongated, 
after  its  exit  from  the  pollen  grain;  If  the  pollen  grain 
be  put  in  the  staining  fluid  without  being  crushed,  the  nu- 
clei will  be  seen  in  their  natural  position,  the  vermiform 
one  somewhat  more  deeply  stained  than  the  other.  The 
rest  of  the  grain  will  remain  uncolored.  If  the  pollen 
grain  be  crushed  in  a  drop  of  water  to  which  has  been 
added  a  solution  of  potassium  iodide  of  iodine,  numerous 
small  blue  starch  granules  will  be  seen  among  the  yellow- 
brown  contents.  (3) 

If  we  now  return  to  the  3^oung  flower-bud  and  take  one 
about  6  mm.  long  and  crush  the  anther  in  water,  we  shall 
find  that  part  of  the  grains  have  but  one  nucleus  and  others 
tw^o,  l3ing  close  together  as  in  Fig.  103,  G.  The  two  nu- 
clei are  separated  by  a  wall  which  incloses  one  of  them, 
together  Avith  a  little  protoplasm.  This  flat  cell  which  is 
almost  circular  in  form  always  lies  on  the  flat  side  of  the 
pollen  grain  where  the  fold  in  the  membrane  is  afterwards 
found.  In  a  somewhat  older  bud  this  cell  is  found  to  be 
separated  from  the  wall  of  the  pollen  grain  and  lies  free 
in  the  contents  of  the  graiu. 


312  POLLEX    OF    CEXOTHEEA. 

The  pollen  gniins  have  here  become  longer  and  corres- 
pondingly slender  and  pointed  at  the  ends.  With  the  ex- 
ception of  the  two  ends  they  are  filled  with  this  nnclei. 
(4).  In  nearly  ripe  pollen  grains  the  definite  demarca- 
tions of  the  nuclei  disappear  and  they  lie  free  in  the  grain 
more  or  less  elongated  in  a  vermiform  shape. 

In  comparison  with  the  gynmosperms,  to  which  they  lie 
very  near,  we  should  hold  the  small  cells  to  be  vegetative  ; 
but  really  it  is  the  generative  cells  Avith  their  highly  stain- 
able  nucleus  which  are  concerned  in  the  fertilization.  The 
difference  in  the  staining  quality  of  vegetative  and  gener- 
ative nuclei  is  far  more  striking  than  in  Tradescantia.  We 
may  make  the  above  described  examinations,  in  the  case  of 
the  younger  pollen  grains  in  pure  water,  but  in  the  older 
stages  we  must  use  meth}l  green  or  iodine  green  with 
acetic  acid.  The  species  of  Lucojum  act  quite  like  the 
Tradescantia. 

If  one  opens  a  bud  of  OEnothera  biennis  which  is  about 
ready  to  blossom,  he  will  find  that  the  anthers  are  already 
open  and  the  pollen  escaped.  Afterwards  viscous  fibres 
are  seen  between  the  anthers.  Putting  one  of  these  on  a 
slide,  it  appears  under  the  microscope  an  extremely  del- 
icate thread  partly  stretched  out  straight  and  in  part  wavy. 
The  pollen  grains  wdien  dry  are  untransparent  but  their 
triangular  form  is  apparent.  In  water  and  with  a  higher 
power  we  see  that  they  are  flattened,  equilateral  triangu- 
lar bodies  with  warty  projecting  corners.  At  the  base  of 
each  of  these  warts  is  to  be  seen  a  ring-shaped  thickening  of 
the  pollen  membrane.  The  contents  of  the  ripe  pollen  grain 
are  tinely  granular,  and  the  two  nuclei  are  seen  with  great 
difficulty.  The  pol  len  membrane  is  stained  a  red-brown  with 
sulphuric  acid.  The  acid  causes  a  very  thin  yellow  layer 
to  be  lifted  up  upon  the  body  of  the  grain,  forming  folds 
from  an  inner,  thicker,  red-brown  la3^er.  Both  membranes 
are  united  in  the  walls  of  the  warts.     From  the  lateral  walls 


POLLEN    OF    ALTH^A.  313 

of  the  warts  fine  teeth  project  toward  the  inside  so  that 
these  walls  appear  to  be  porous.  The  apex  of  the  wart  is 
dissolved  by  the  acid.  The  fine  filaments  which  connect 
the  pollen  grains  are  insoluble  in  water,  alcohol,  potash  lye 
and  sulphuric  acid.  In  25%  chromic  acid,  the  pollen  mem- 
brane dissolves,  the  strongly  cutinized  parts  rather  before 
the  others,  the  latter  being  the  caps  upon  the  projecting 
warts,  which  remain  colorless  and  swollen.  These  finally 
dissolve  and  even  the  viscid  filaments  between  the  pollen 
grains  cannot  withstand  chromic  acid.  A  pollen  grain 
taken  from  the  stigma  of  an  old  flower  will  show  the  pollen 
tubes  already  grown  out,  commonly  from  only  one  wart, 
but  if  from  another  also,  then  only  just  outside  the  latter. 
The  membrane  of  the  pollen  tube  passes  into  the  lateral 
"walls  of  the  wart.  An  intine  layer  as  distinguished  from 
the  outer  membrane  does  not  occur  (5).  Instead  of 
(Enothera  one  may  use  Epilohium  or  Fuchsia. 

AVe  will  now  examine  some  peculiarly  formed  pollen 
grains.  Those  of  the  Malvacece  are  of  extraordinarily 
large  size.  The  pollen  of  Althoea  rosea  in  Avater  are 
globular,  untransparent  and  beset  with  colorless  spines. 
They  become  beautifully  transparent  in  carbolic  acid,  and 
in  chloralhydrate,  less  so  in  oil  of  cloves,  still  less  in 
lemon  oil.  The  best  preparation  is  with  carbolic  acid  and 
so  we  will  use  that.  A  superficial  vieAv  shows  us  that  the 
colorless  pollen  membrane  is  beset  with  large  pointed 
spines  at  nearly  uniform  intervals.  Between  those  are 
others  ;  short,  blunt  and  of  varying  thickness.  Eegularly 
distributed  circular  openings  appear  in  the  membrane. 
The  surfiice  of  the  membrane  is  finely  dotted.  The  con- 
tents of  the  grain  are  imiformly  finely  granular,  and  the 
nucleus  is  made  out  with  much  difiiculty.  An  optical 
transection  of  the  grain  show^s  us  clearly  the  form  of  the 


314  POLLEN    OF    THE    PUMPKIN. 

large  and  small  spines  and  the  canals  which  perforate  the 
membrane.  An  extraordinarily  delicate  but  really  exist- 
ing intine  may  be  traced  only  as  the  outline  of  the  con- 
tents. It  is  papillately  arched  a  little  in  the  canals  of  the 
exine.  In  concentrated  sulphuric  acid  the  exine  soon 
stains  a  red-brown  and  shows  its  structure  then  very  dis- 
tinctly. 

Most  of  the  pollen  grains  of  the  Malvaceoi  act  like  those 
of  AllJicea.  In  Malva  crispa,  a  frequently  cultivated  spe- 
cies, the  pollen  grains  are  like  those  of  AliJicea,  except 
that  the  spines  are  all  alike.  Between  the  spines  are  dis- 
tributed the  openings  in  the  membrane,  the  rest  of  which 
appears  finely  dotted. 

The  large  pollen  grains  of  the  Cucurbita  species  have 
long  received  special  distinction  on  account  of  the  cover 
which  closes  the  opening  in  the  exine.  In  water,  yellow 
oil  drops  exude  from  the  surface  of  the  exine,  the  grain 
soon  l)ecomes  empty  of  its  contents  and  tlie  sti'ucture  of 
the  membrane  may  then  be  distinctly  seen.  The  exine  is 
beset  at  regular  intervals  with  large,  and  between  these 
with  many  small  spines.  The  openings  are  round.  The 
cover  is  lifted  up  on  one  side  or  all  around,  l)y  the  papil- 
lately arched  intine.  It  has  the  structure  of  the  adjoining 
exine  and  bears  one  or  more  spines.  Very  good  prepara- 
tions are  got  by  using  lemon  oil,  less  serviceable  ones  in 
oil  of  cloves,  but  those  in  chloral  hydrate  and  those  in 
car1)olic  acid  are  preferable.  In  each  case  that  medium 
most  suitable  for  clarifying  it  is  to  be  sought  for.  By  op- 
tical transection  in  lemon  oil  or  chloral  hydrate  prepara- 
tions we  are  able  to  demonstrate  the  position  of  the  cover 
within  the  exine,  and  find  it  widened  inward  somewhat 
towards  the  base.  Under  the  cover  the  swellino;s  of  the 
intine  may  be  seen.     The  oil  drops  on  the  exine  are  col- 


DEVELOPMENT    OF   THE  POLLEN   TUBE.  315 

ored  blue  with  sulphuric  acitl.  The  exine  becomes  grad- 
ually browu.  The  cover  is  pushed  off  by  the  swelling 
contents.  In  25%  chromic  acid  the  whole  pollen  mem- 
brane is  soon  dissolved,  but  the  intine  withstands  it  longest 
and  is,  at  the  moment  where  the  exine  disappears,  clearly 
discernible  as  a  greatl}^  swollen  homogeneous  membrane. 
The  pollen  grain  has  previously  become  empty,  which  fjx- 
cilitates  the  examination  of  the  intine.  In  sulphuric 
acid,  on  the  contrary,  the  intine  is  immediately  dissolved, 
while  the  exine  remains  and  the  exuding  contents  of  the 
grain  gradually  assume  a  rosy  tint  as  in  other  cases. 

Of  compound  pollen  grains,  which  occur  both  in  mono- 
and  dicotyledons,  we  will  look  at  those  of  CaUuna  vulga- 
ris first.  The  grains  are  united  into  fours  and  mostly 
tetrahedrically  grouped.  The  pollen  membrane  shows  but 
small  protuberances  and  mostly  but  three  openings  to  each 
grain.  The  species  of  Erica,  Azalia  and  Uliododendron 
are  essentially  the  same  as  those  of  Calluna.  In  the  Aca- 
cia &t^qc\^q,  especially  in  Mimosa  (6),  the  pollen  grains 
form  groups  of  four,  eight,  twelve,  and  sixteen,  or  even 
more. 

In  a  sugar  solution  of  from  3  to  30%  which  contains 
1.5%  gelatin,  most  pollen  grains  will  put  out  three  tubes, 
in  which  the  streaming  of  the  protoplasm  may  be  beauti- 
fully *eeu.  The  formation  of  pollen  tubes  takes  place 
rapidly  and  surely  in  a  5%  solution  of  sugar  with  1.5% 
gelatin  with  pollen  grains  from  the  Peonia,  Slaphylea  and 
also  when  they  are  taken  from  a  freshly  opened  flower  of 
Tradescantia.  The  most  favorable  objects  are  furnished 
by  the  species  of  Latliyrus  in  15%  sugar  solution  with 
1.5%  gelatin.  The  solution  must  be  freshly  prepared  and 
the  experiment  is  best  made  in  a  hanging  drop  in  a  moist 
chamber.     See  page  230. 


316  LITERATURE  OF  THE  LESSON. 

Notes. 

(1)  For  staraeus  aud  pollen,  see  v.  Mohl,  Ueber  deu  Bau  und  die 
Formen  der  Pollenkörner,  1834;  Fritsclie,  Ueber  den  Pollen,  Mem. 
de  sav.  Strang,  1836 ;  Naegeli,  Zur  Entwickelungsg.  d.  Poll,  ble  den 
Phan.,  1842;  Schacht,  Jahrb.  f.  wiss.  Bot.  Bd.  ii,  p.l09;  Warming  in 
Hanstein's  bot.  Abh.  Bd.  ii,  Heft  ii;  Strasburger,  Befr.  u.  Zellth.,  p.  15 
und  Bau  der  Zellhäute,  p.  86;  Elfving,  Jeu.  Zeitschr.  f.  Naturw.  Bd, 
XIII,  p.  1;  Goebel,  Grundz.  d.  Syst.,  etc.,  p.  398;  Luerssen,  üruudz. 
d.  Bot.,  III  Auf.,  p.  359;  Med.  Pharm.  Bot.,  Bd.  ii,  p.  198;  Prantl, 
Lehrb.  d.  Bot.  iv  Aufl.,  p.  192.  In  the  above  quoted  works  is  the  rest 
of  the  literature. 

(2)  Sachs,  bot.  Ztg.,  1862,  p.  242. 

(3)  "Warming  in  Hanstein's  Bot.  Abh.  Bd.  ii,  Heft  ii;  Goebel, 
Grundzüge,  p.  409. 

(4)  See  also  Elfviug,  Jauaische  Zeitschr.  f.  Naturwiss.  Bd.  xiii, 
p.  12. 

(5)  Strasburger,  Bau  d.  Zellh.  p.  95.  There  also  the  history  of 
its  development. 

(6)  Rosanoff,  Jahrb.  f.  wiss.  Bot.  Bd.  iv,  p.  441 ;  Engler,  the  same 
periodical  Bd.  x,  p.  277.     There  also  the  literature. 


LESSON  XXIX. 
The  Gyneceum  of  the  Angiosperms. 

We  will  now  take  a  general  survey  of  the  structure  of 
the  ovary  (1)  using  the  Delphinium  ajacis  or  garden 
larkspur  for  our  purpose.  Take  an  old  flower  from  which 
the  petals  and  stamens  may  be  easily  removed,  and  notice 
the  three  pistils  which  remain  standing  in  the  middle. 
We  shall  see  first  the  green  swollen  part,  the  ovary,  the 
slender  rose-colored  portion  into  which  the  ovary  narrows 
itself,  the  style.  This  tinally  ends  in  the  stigma  which 
is  not  in  this  case  especially  developed 
but  simply  terminates  the  style.  Make 
a  transection  through  the  three  ovaries, 
and  examine  with  a  low  power  adding 
a  little  potash  lye.  The  transection 
shows  us  for  each  ovary  a  single  cavity, 
Fig.  104.     Apparently  it  is  a   single 

iruit-leat    or    carpel    which    torms    each     ajads.     Transection    of 

ovary.  The  carpellary-leaf  is  folded  to-   °''-'''-\-  ";  """'^'^ ^''■^"=  "'• 

•^       _  '■  •'  ^  vascular  bundle ;  7;,  pla- 

gether  inwardly  and  its  edges  o;rown  centa;  s,  embryo  seed. 
fast  to  each  other  forming  the  "ventral 
seam,"  so  called,  which  we  find  in  the  middle  of  the  ovary 
on  the  side  which  faces  the  centre  of  the  flower.  An 
ovary  formed  of  one  carpellary-leaf  is  monocarpous,  but 
when  several  such  ovaries  are  united  in  one,  as  in  this  case, 
the  flower  is  said  to  be  polycarpous.  The  ovaries  are  in 
this  example  free  to  their  base  where  they  are  inserted  on 
the  receptacle  and  are  called  superior.  The  whole  female 
generative  apparatus,  whether  it  consists  of  one  or  more 
pistils,  is  designated  the  gy necium. 

(317) 


# 


318  STRUCTURE  OF  THE  OVARY. 

Our  transection  clearly  shows  the  furrow  on  the  ventral 
side,  and  by  the  use  of  a  higher  magnificat  ion  w^e  can  eas- 
ily trace  the  epidermis  at  the  outside  at  this  place  through 
the  whole  thickness  of  the  wall  and  see  that  it  is  continued 
into  the  epidermis  of  the  cavity  of  the  ovary.  Even  sto- 
mata  are  found  in  this  inner  epidermis.  The  ovary  walls 
are  penetrated  by  a  number  of  vascular  bundles  most  of 
which  show  on  the  backside,  but  some  near  the  edges  of  the 
carpellary  leaf  on  the  ventral  side.  The  edges  of  the  car- 
pellar}'  leaves  are  somewhat  swollen,  and,  in  the  cavity  of 
the  fruit  receptacle,  is  formed  into  a  phicenta,^^.  From  this 
the  ovules,  s,  originate  in  two  series,  corresponding  to  the 
number  of  the  placentae.  Vie  shall  give  particular  atten- 
tion to  the  ovule  later  on,  and  to  this  end  lay  aside  our 
preparation. 

In  the  blossom  of  Butomus  umbellalus  are  six  ovaries. 
But  these  ovaries  are  free  only  in  their  upper  halves,  while 
they  are  grown  together  laterally  below  and  cannot  be 
separated  without  injury.  The  pistil  is  ver}^  short  and 
the  upper  edge  of  it  is  the  stigma.  We  must  prepare 
transections  both  of  the  free  and  the  united  parts  of  the 
ovary.  In  those  of  the  upper  part  we  may  easily  distin- 
guish the  carpellary  leaves  as  in  the  Delpldnium,  but  in  the 
sections  from  below  the}' cannot  be  isolated  intact  laterally 
from  each  other.  lu  the  Butomus  we  have  an  interme- 
diate form  between  the  polycarpous  and  monocarpous  flow- 
ers, and  this  will  serve  us  as  an  example  of  a  compound 
ovary  formed  out  of  more  than  one  carpellary  leaf.  A 
marked  peculiarity'  of  the  Butomus  is  seen  in  the  fact  that 
the  ovules  do  not  spring  alone  from  the  edges  of  the  carpel- 
lary leaves,  but  rather  from  their  midd  le  and  from  their  whole 
inner  surface.  The  whole  wall  of  the  ovary  is  beset  with 
them  and  acts  as  a  parietal  placenta.  At  the  point  of  in- 
sertion of  each  ovule  a  fine  vascular  bundle  may  be  seen, 


STRUCTURE    OF   THE    OVARY.  319 

which  provides  for  the   ovule.     These  are  branches  of 
larger  bundles  lying  deeper  in  the  tissue. 

The  carpellary  leaves  of  the  Liliacece  are  superior. 
Take  for  our  investigation  a  tulip,  hyacinth,  a  lily  or  a 
Hemei'ocallis.  In  the  tulip  the  three  stigmas  rest  on  the 
ovary  without  a  style.  In  the  hyacinth  the  style  is  short, 
the  stigma  small,  dark,  divided  into  three  parts.  In  the 
lily  the  style  is  long,  the  stigma  three-parted.  In  Hemero- 
callis  the  style  is  ver\'  long  with  a  three-parted,  still  very 
small  stigma.  A  transection  will  show  us  a  compound 
ovary  formed  of  three  closed  carpellary  leaves  grown  to- 
gether. No  boundary  between  the  parts  either  at  the  side 
or  in  the  middle  is  to  be  recos^nized  here.  A  continuous 
epidermis  covers  the  Avhole  organ.  Three  carpellary 
leaves  form  a  compound  ovary  with  three  cells.  Each 
of  the  three  carpels  which  form  this  ovary  has  two  series 
of  ovules,  lying  along  its  two  edges.  The  placenta  there- 
fore lies  in  the  inner  angle  of  the  ovarj^  cell.  The  pla- 
centa is  therefore  marginal  as  in  Delphinium,  and  since  it 
springs  from  the  angle  of  the  ovary  which  turns  toward 
the  middle  it  is  called  "central."  A  transection  of  the 
pistil  of  Hemerocallis  shows  us  a  three-cornered  style,  in 
which  toward  the  three  edges  are  distributed  three  vascu- 
lar bundles.  A  longitudinal  section  of  the  pistil  which 
cuts  the  stigma  will  show  that  the  latter  is  developed  into 
long  papillte  on  its  upper  surface.  This  is  the  most  com- 
mon appearance  of  the  stigma.  But  in  the  Hemerocallis 
we  find  the  cuticle  of  the  papillt©  raised  up  by  the  pressure 
of  muciluge  formed  beneath.  The  cuticle  is  spirally 
striped  and  conformalily  to  this  the  elevations  follow  a 
spiral  line.  The  cuticle  Avill  finally  be  separated  from  the 
inner  layer  and  eventually  removed  from  the  papillte.  The 
other  Liliacece  might  likewise  show  us  a  holloAV  style,  but 
in  most  flowers  it  is  solid,   filled  with  cells  with  swollen 


320  STRUCTURE    OF   THE    OVARY. 

side  walls,  or  with  those  entering  from  the  lateral  tissue, 
between  which  the  pollen  tube  can  easily  grow  down- 
wards. 

The  Primularia  species  have  a  superior  ovary.  They 
are  dimorphic,  that  is  they  show  short  and  long  styled 
ovaries  and  stamens  inserted  above  and  below  on  the  co- 
rolla. A  median  longitudinal  section  through  the  ovary 
shows  us  that  the  axis  of  the  flower  continues  into  the 
cavity  of  the  ovary  and  expands  into  the  shape  of  a  toad- 
stool. In  the  middle  this  expansion  rises  into  the  style  of 
the  pistil  in  a  papillate  form  and  the  whole  upper  surface 
is  beset  with  ovules.  We  have  in  this  case  a  free  central 
placenta.  The  walls  of  the  ovary  nowhere  join  this  pla- 
centa, as  is  seen  by  a  transection  in  which  these  walls  ap- 
pear as  a  free  ring  about  the  central  placenta.  The  point 
of  juncture  or  suture  is  not  visible  in  this  ring:  so,  in  or- 
der to  determine  the  number  of  carpellary  leaves  which  go 
to  form  the  ovary,  we  must  refer  to  the  number  of  the 
other  parts  of  the  flower  and  to  the  circumstance  that  in 
many  Primidaceoe  the  seed  capsule  opens  at  the  top  with 
five  teeth,  and  thus  conclude  that  there  are  five.  In 
Primula  itself  the  number  of  teeth  with  which  the  capsule 
opens  is  indefinite.  Instead  of  the  Primula  we  may  take 
species  of  the  Lysimachia  or  Anagallis  for  an  investiga- 
tion, as  they  have  all  their  ovules  on  a  free  central  pla- 
centa. 

We  shall  now  take  an  inferior  ovary — that  of  the  Epi- 
jpactis  palustris  or  some  other  orchid.  The  brown  ovary 
lies  beneath  the  other  floral  parts.  We  Avill  select  for 
our  section  a  young  fruit  over  which  the  floral  leaves 
have  already  begun  to  be  brown.  The  transection  is  very 
instructive.  It  shows  us  a  simple  ovary  which  bears  on 
the  walls  at  equidistant  points  three  double  pairs  of 
placentae.     The  placenta  divides  repeatedly  on  the  edge 


OVARY    OF    THE    ORCHID.  321 

and  bears  a  large  number  of  ovules.  Upon  the  outside 
of  the  ovary  arc  six  projecting  ril)s,  three  of  them  corre- 
sponding to  the  place  of  insertion  of  the  placentai  within 
and  the  other  three,  especially  large  ones,  alternate  with 
these  places.  Each  rib  is  furnished  with  a  Vascular  bun- 
dle or  with  a  complexity  of  them,  and  besides  this  a  small 
one  at  the  place  of  separation  of  two  placentie.  If  we 
were  to  bestow  no  thought  upon  it,  this  section  would 
seem  to  agree  perfectly  with  one  made  from  a  superior 
ovary,  the  ovary  would  appear  to  be  formed  from  three 
carpellary  leaves  and  the  pairs  of  placentae  to  be  produced 
on  the  united  edges  of  two  such  adjacent  leaves,  and  the 
three  ribs  which  alternate  with  the  places  of  insertion  of 
the  placenti^ä  would  be  held  to  be  the  midribs  of  the  leaves. 
But  as  this  is  an  inferior  ovary  the  case  is  far  less  simple. 
TVe  may  suppose  either  that  the  inferior  ovary  is  formed 
from  an  excavated  floral  axis  terminated  above  with  car- 
pellary leaves  and  that  from  the  latter  the  placent»  con- 
tinue downward  into  the  excavation,  or  we  may  suppose 
that  the  carpellary  leaves  are  grown  to  the  hollow  floral 
axis ;  consequently  the  outer  part  of  the  wall  of  the  in- 
ferior ovary  belongs  to  the  stem,  and  the  inner  part  to  the 
carpellary  leaves.  The  latter  supposition  is  decidedly  to 
be  preferred,  but  it  has  no  other  than  a  phylogenetic  value, 
that  is  to  say,  we  represent  to  ourselves  that  in  the  course 
of  time  the  inferior  ovary  is  thus  produced.  But  in  reality 
in  the  object  itself  there  is  no  moment  in  its  developmental 
history  when  it  anatomically  answers  to  this  supposition. 
AVe  must  be  content,  therefore,  to  demonstrate  that  the 
structure  of  this  inferior  ovary  is  not  essentially  different 
from  that  of  a  simple,  polymerous,  superior  ovar3\  By 
examining  a  ripe  fruit  capsule  of  Epipactus^  we  shall  find 
that,  as  in  most  orchids,  the  wall  splits  into  six  longitud- 
inal openings,  the  segments  between  remaining  united  at 

21 


322 


OVULE    OF    ACONITUM. 


the  top  and  bottom  of  the  ovary.  Three  of  them  are 
broad  and  fertile  and  three  narrow  and  sterile,  the  Litter 
corresponding  to  the  median  ribs  which  we  saw  in  the 
transection  of  the  ovary,  forming  the  intermediate  sections. 
The  three  fertile  segments  bear  the  placentae. 

We  shall  next  nndertake  to  investigate  the  structure  of 
the  ovule  and  the  process  of  its  fertilization  in  the  angi- 
osperms.  In  order  to  get  a  view  of  the  separate  parts 
of  the  ovule,  make  a  transection  of  the  ovary  of  Aconitum 

Napellus  or  of  some  other  spe- 
cies of  this  genus.  Take  a 
flower,  just  turning  blue,  strip 
otF  the  floral  parts  and  make  a 
section  of  the  three  ovaries  at 
once,  care  being  taken  that  the 
section  is  really  at  right  angles 
with  the  axis  of  the  ovary. 
Make  a  considerable  number  of 
sections,  so  as  to  be  sure  to 
have  one  which  cuts  the  ovule 
at  the  right  place.  Look  them 
over  and  select  the  best  section, 
and  in  case  it  is  not  thin  enough 
a  little  potash  lye  will  help 
make  it  transparent.  The  im- 
age will  be  almost  identical  with  that  of  the  previously 
examined  Delphinium.  Still  the  structure  of  the  envelope 
of  the  ovule  is  a  little  diflerent,  and  this  ditterence  gives 
it  the  preference.  The  ovary  is  monomerous.  The  ovules 
spring  from  a  placenta  formed  on  the  infolded  edge  of 
the  carpellary  leaves.  They  are  inserted  with  a  small  style 
or  funiculus,  y,  whose  free  part  is  quite  short,  but  the  rest 
of  it  is  grown  fest  to  the  body  of  the  ovule  and  forms  the 
so-called  raphe,  r.     In  the  body  of  the  ovule  we  distin- 


FlG.  105.  Aconitum  Napelhis. 
Xiongitiidinal  section  of  ovule;/, 
funiculus;  r,  raphe;  ti,  vascular 
bundle  of  funiculus;  ie  and  ii, 
outer  and  inner  integument;  n,  nu- 
cellus;  ch,  chalaza;  e,  embryo  sac; 
a,  antipodal  cells;  o,  egg;  nc,  nu- 
cleus of  the  embryo  sac  ;  m,  micro- 
pyle;  or,  wall  of  the  ovary.     X  53. 


STRUCTURE    OF   OVULE.  323 

gnish,  first  of  uU,  the  inner  cone-shaped  muss  of  tissue, 
the  ovule  nucleus,  or  nucellus,  n.  This  corresponds  to 
the  macrospore  of  the  vascular  cryptogams.  It  is  sur- 
rounded by  two  integuments  :  an  inner,  ^^,  and  an  outer, 
ie.  The  inner  one  is  developed  all  around  down  to  the 
base.  The  outer  one  fails  on  the  side  of  the  funiculus, 
with  which  it  is  laterally  connected.  Between  the  upper 
edges  of  the  inner  integument  is  a  free  opening  or  canal, 
the  micropyle,  m,  down  to  the  nucellus.  In  the  funiculus 
a  vascular  bundle  coming  from  the  placenta  may  be  traced, 
sometimes,  but  not  always,  quite  down  to  the  base  of  the 
nucellus.  At  the  base  of  the  nucellus  is  a  mass  of  clear 
tissue  called  the  base  of  the  ovule,  or  chalaza,  ch.  In  the 
axis  of  the  nucellus  is  the  lar<>:e  cavitv-forming  cell  of  the 
embryo  sac,  e.  At  the  base  of  this  are  some  spherical  cells, 
which  in  the  Aconitum  and  in  Ranunculacem  generally  are 
strongly  developed  ;  they  are  the  so-called  antipodal  cells, 
a.  In  specially  favorable  cases  they  may  be  seen  to  oc- 
cur in  threes.  At  the  apex  of  the  embryo  sac  a  small 
cell  may  be  made  out — but  only  in  an  exactly  median  sec- 
tion—  which  is  the  ovum  cell,  o.  The  whole  ovule  is 
anatropic  or  recurrent  because  the  body  of  the  ovule  is  not 
an  elongation  of  the  funiculus,  but  is  folded  back  upon  it 
and  partly  surrounded  by  it  and  grown  fast  to  it,  the  mi- 
cropyle being  turned  towards  the  base  of  the  funiculus. 
This  form  of  ovule  largely  prevails  in  angiosperms.  If 
we  now  compare  this  preparation  with  that  o^  Deljplilnium 
we  shall  find  that  the  structure  is  almost  identical  with  it, 
the  only  difference  being  that  in  the  DeljyJdnium  the  two 
inteo-uments  of  the  ovule  are  united  into  one. 

In  order  to  make  a  good  section  of  the  ovule  in  the  or- 
dinary way  between  the  thumb  and  finger  we  must  re- 
move it  from  the  ovary.  If  it  is  rightly  placed  between 
the  thumb  and  finger  we  may  obtain  a  median  view  this 


324  MAKING    SECTIONS    OF    THE    OVULE. 

way  sooner  than  by  any  other.  But  we  may  with  advan- 
tage embed  the  ovule  in  glycerine  jelly  or  in  collodion  be- 
fore cutting.  The  glycerine  jelly  must  be  relatively  stiff, 
that  is,  contain  considerable  gelatine.  Only  alcohol  ma- 
terial can  be  embedded  in  collodion.  Pour  the  collodion 
solution  in  a  little  box  made  of  Avriting  paper  and  lay  the 
ovule  in  it.  It  must  stand  in  the  air  till  it  stiffens  so  that 
it  will  not  run,  then  put  it  in  60  to  90  %  alcohol.  Here 
it  will,  in  the  course  of  a  few  hours,  become  of  the  consist- 
ency of  gristle  and  be  transparent ;  cut  through  the  object 
and  the  collodion  together,  and  transfer  the  section  with- 
out removing  the  collodion  to  glycerine  or  glycerine  jelly. 
If  one  has  got  his  collodion  in  the  form  of  tablets  he  must 
dissolve  it  in  a  solution  of  equal  parts  ether  and  absolute 
alcohol.  In  order  to  make  the  ovule  visible  in  the  em- 
bedding medium  it  may  be  first  stained  in  an  aqueous  so- 
lution of  ha^niatoxylin.  But  the  water  must  be  afterwards 
removed  from  the  object  by  the  use  of  absolute  alcohol  be- 
fore it  can  be  embedded  in  the  collodion. 

For  the  study  of  the  interior  of  the  embryo  sac  we  will 
take  the  Monotropa  Hypo^itys,  or  false  beech  drops  (2), 
the  pale  yellow  plant  common  in  pine  forests.  It  is  such 
a  very  favorable  object  for  our  purpose  that  we  should 
spare  no  pains  to  obtain  it.  It  blossoms  in  June  and  July 
and  must  be  examined  fresh  since  alcohol  turns  it  dark 
brown  and  makes  it  opaque.  It  may  be  kept  for  a  long 
time  in  a  glass  of  water.  Species  of  Pyrola  answer  the 
same  purpose,  only  that  the  ovules  are  smaller.  A  tran- 
section of  the  superior  ovary  shows  it  to  be  fom'-celled. 
The  placentae  are  much  swollen  and  bear  on  their  surface 
small,  very  numerous,  closel}'  compacted  ovules.  The  two 
halves  of  the  placentae  in  each  compartment  are  widely 
separated  by  radial  lines.  In  the  upper  part  of  the  ovary 
these  lines  reach  the  middle  and  touch  each  other.     We 


OVULE  OF  FALSE  BEECH  DROP. 


325 


now  see  four  stout  pairs  of  placentoe  fixed  to  the  middle 
of  the  division  Avails  which  heloug  to  each  two  neighboring 
compartments,  the  pairs  being  easil}'  separated  with  the 
needle.  Remove  the  ovules  from  the  open  placenta  with 
a  needle  and  put  them  in  pure  water  or  a  3%  solution  of 
sugar  in  which  the  ovules  will  keep  a  long  time.  If  we 
get  our  material  from  an  old  flower  in  which  the  stamens 


Fig.  106.  Monotropa  FTi/popitys.  A,  a  whole  ovule;  /,  the  funiciilns;  i,  the  iu- 
tegiimeut;  B  and  C,  the  whole  embryo  sac;  s,  synergUla;;  o,  egg;  n,  nucleus  of  em- 
bryo sac ;  D  and  /i,  the  upper  part  of  the  embryo  sac.  In  E,  is  the  first  division  for 
the  Ibrmatiou  of  tlie  endosperm.  A  X  210-    JB  to  E.  X.  600. 

have  already  discharged  their  pollen,  we  shall  find  the 
ovules  ripe,  but  in  part  already  fertilized,  and  in  part  not. 
Between  the  ovules  we  shall  find  many  pieces  of  pollen 
tubes.  The  ovule  ripe  for  fertilization  is  seen  as  in  Fig. 
106,  A..  It  is  transparent  and  may  be  seen  in  optical  sec- 
tions. It  is  anatropic  and  has  but  one  integument,  i. 
The  whole  interior  of  the  ovule  is  filled  with  .the  embryo 


326  FERTILIZATION   OF   THE    OVULE. 

sac,  the  niicellus  being  suppressed  by  the  growth  of  the  em- 
bryo sac.  We  will  assume  that  the  three  cells  of  the  apex 
of  the  embryo  sac  are  clearly  seen.  These  three  cells  form 
the  egg  apparatus.  They  are  not  of  equal  value.  The  two 
upper  ones  are  helpers  or  synergidae,  Fig.  106,  B;  the 
lower  one  is  the  true  egg,  o.  The  synergidte,  as  may  be 
easily  seen,  have  large  vacuoles  in  their  lower  part  and 
are  filled  above  with  protoplasm  and  at  that  point  have 
their  nuclei.  The  egg,  on  the  contrary,  lies  between  the 
principal  mass  of  the  cell  plasma  and  cell  nucleus  and 
above  the  cell  cavity.  We  may  not  always  see  both  syn- 
ergidi«,  as  one  may  cover  up  the  other,  Fig.  106,  C.  At 
the  base  of  the  embiyo,  sac  one  may  see  the  three  antipo- 
dal cells.  In  the  interior  of  the  embryo  sac,  one  may  find 
in  most  cases  a  cell  nucleus,  Fig.  106,  A.  Still  in  other 
cases  there  are  two  nuclei,  Fig.  106,  B,  or  one  cell  nucleus 
with  two  nucleus  bodies.  Fig.  106,  C,  and  we  conclude 
that  in  the  end  the  cell  nucleus  is  made  from  the  union 
of  two  nuclei.  Ovules,  whose  fertilization  has  begun, 
show  the  fact  in  the  changes  which  have  taken  place  in 
the  synergidte.  One  or  both  of  them  appear  strangely 
refractive.  A  pollen  tube  may  also  be  seen  penetrating 
the  embryo  sac,  or  at  least,  within  the  micropyle,  or  a  piece 
of  it  projecting  from  the  micropyle,  torn  away  in  the  prep- 
aration. But,  if  the  pollen  tube  has  penetrated  to  the 
synergidae,  the  plasma  from  it  will  be  thrown  in  between 
these  cells  upon  the  ovum  itself.  By  careful  examination, 
it  Avill  be  seen  that  an  ovum  which  lies  near  these  changed 
S3aiergidje  has  two  nuclei :  one  large,  the  original  nucleus 
of  the  ovum,  and  a  much  smaller  one  which  is  the  sperm 
nucleus  from  the  pollen  tube.  Fig.  106,  D.  The  latter  soon 
increases  in  size.  It  one  finds  the  ovule  at  the  moment  of 
copulation  between  the  egg  nucleus  and  the  sperm  nucleus 
he  will  see  but  one  germinal  nucleus  with  two  nuclei  of  un- 


DEVELOPMENT    OF    FERTILIZED    OVULE.  327 

like  size,  Fig.  106,  E,  of  which  the  smaller  is  the  spermatic 
nucleus.  At  last  the  germinal  nucleus  will  have  but  oue 
nucleolus.  While  the  fertilizatiou  of  the  ovum  is  going  ou 
the  strongly  refractive  sul)stance  in  the  synergicÜB  cells  is 
lessened,  apparently  being  used  up  in  nourishing  the  ovum. 
At  the  same  time  with  these  changes  in  the  egg  appara- 
tus, the  endosperm  has  begun  to  form  in  the  cavity  of 
the  embryo  sac,  by  the  development  of  division  walls  in 
the  sac  itself,  in  this  case  by  direct  cell-division  ;  but  in 
other  cases,  as  freqnently  or  more  often,  the  embryo  sac 
nucleus  and  its  derivatives  freely  divide  first,  followed 
later  by  the  formation  of  division  walls  between  the  nuclei. 
This  process,  as  it  commonly  takes  place,  is  accompanied 
by  a  gradual  but  not  considerable  increase  in  size.  When, 
on  the  contrary,  the  embryo  sac  rapidly  grows  afterthe  com- 
pletion of  the  fertilization  of  the  ovum,  it  takes  place  first 
by  nucleus  and  not  l>y  cell  division,  the  cell-building  com- 
ing later  when  the  emliryo  sac  is  nearly  full  grown.  In 
consequence  of  the  lertilization,  the  ovum  has  taken  on  a 
delicate  cellulose  membrane  and  soon  beofins  to  elongate 
tube-like  and  after  a  time  penetrates  the  endosperm  body 
with  its  apex,  where  it  forms  an  embryo  of  a  few  cells. 
AYe  have  examined  the  embryo  seed  only  in  pure  water  or 
in  sugar  solution.  If  we  would  have  the  nucleus  espec- 
ially distinct,  we  should  examine  it  in  a  2%  solution  of 
acetic  acid.  We  thei'eby  fix  the  nucleus  and  make  it  very 
sharply  distinct  and  also  preserve  it  in  that  state  of  self- 
division  in  which  it  was  at  that  moment.  Staining  media 
are  not  recommended  since  they  stain  also  the  integument 
and  thereby  hinder  the  examination  of  the  interior  of  the 
nucleus. 

Instead  of  Mo7iotropa  the  orchids  (3)  would  serve  us. 
Fertilization  takes  place  a  long  time  after  the  discharge  of 
the  pollen  in  the  already  greatly  swollen   ovary.      Cut 


328 


OVULE  OF  ORCHIS  PALLEXS. 


away  the  ovary  and  remove  an  ovule  from  the  placenta 
with  a  needle  and  transfer  it  to  water  or  a  3  %  solution 
of  sugar.  As  represented  in  Fig.  107,  m,  we  see  that  the 
structure  of  the  ovule  is  much  like  that  of  3Ionotropa, 
except  that  there  are  two  integuments,  and  an  air  cavity  in 
the  vicinity  of  the  chalaza.       The  air  cavity  hinders  the 

observation  since  it  is  tilled  with 
air  which  tinally  works  up  be- 
tween the  integument.  It  ma}'' 
be  taken  out  with  the  air  pump 
or  perhaps  by  a  light  pressure 
upon  the  cover-glass.  In  the 
orchids  the  nucellus  is  quite  sup- 
pressed by  the  embryo  sac.  The 
egg  apparatus,  os,  is  built  like 
that  of  Monotropa,  only  that  the 
ovum  is  less  deeply  inserted. 
The  antipodal  cells  are  not  to 
be  seen,  but  in  their  place  a 
strongly  refractive  substance  in 
which  are  nuclei  very  difficult 
to  make  out. 

It  is  easier  to  trace  the  pol- 
len tube  to  the  synergidae  than 

letters  are  the  same  as  in  the  earlier    in    the   MoilOtvOpa  ;  the    chaUgCS 

which  take  place  in  the  syner- 
gidaj  are  quite  the  same.  We  also  find  again  the  two 
nuclei  in  the  fertilized  ovum.  Endosperms  are  not  usu- 
ally formed. 

In  lack  of  jSlonotroixi  and  orchids  the  transparent 
ovules  of  the  Gesneriacece  (4)  are  to  be  commended  and 
before  all  others  the  large-flowered  Gloxinia  liyhrida.  The 
ovule  with  an  integument  is  so  far  transparent  that  the 
Qgg  apparatus  is  distinctly  visible.     It  shows  the  two  syn- 


FiG.  107.  Orchis  paUens.  Ovule 
ripe  for  feriilizatioii.  os,  egg  appar- 
atus; ii,  le,  inner  and  outer  intefrii- 
nients;  I,  air  cavity.  The  rest  of  the 


FERTILIZATION    OF   TORENIA.  329 

ergldte  and  ovum,  in  this  case  fork-shaped.  Sometimes 
two  ova  appear.  The  embryo  sac  is  swollen  above,  but 
is  suddenly  narrowed  below.  The  antipodal  cells  are  not 
made  out  with  certainty. 

But  one  of  the  most  favorable  plants  for  the  study 
of  fertilization  is  the  Torenia  asiatica  (5).  It  is  cultivated 
in  almost  all  gardens  and  bears  flowers  the  year  around. 
It  is  distinguished  by  having  the  embryo  sac  protrude 
through  the  micropyle  of  the  ovule,  and  so  the  egg  ap- 
paratus comes  into  view,  covered  only  by  the  wall  of  the 
embryo  sac.  The  ovary  is  two-celled,  the  placenta  cen- 
tral, with  many  ovules.  Remove  some  of  the  ovules  and 
examine  in  3  %  sugar  solution.  The  ovules  are  anatropic 
or  more  rightly  somewhat  campylotropic  for  the  embryo 
sac,  and  its  integuments  are  somewhat  bent  in  their  upper 
part,  Fig.  108,  A.  The  funiculus,  /,  is  somewhat  large 
and  the  single  integument  stout.  The  embryo  sac,  e, 
shows  its  upper  end  protruded  through  the  micropyle, 
smaller  and  pointed  and  lying  against  the  funiculus.  By 
means  of  potash  lye,  at  the  outset  of  its  action,  the  em- 
bryo sac  may  be  traced  downwards  into  the  ovule,  where 
it  may  be  seen  to  lie  next  the  integument,  very  slender, 
somewhat  spindle-shaped,  e*,  and  at  the  base  again  nar- 
rowed. Our  preparation  in  sugar  water  shows  the  three 
cells  of  the  egg  apparatus  in  the  apex  of  the  embryo  sac. 
According  to  the  position  of  the  ovule,  will  both  syner- 
gidaB  or  one  be  shown,  as  in  B  or  C ;  m  the  latter  case 
one  covers  the  other. 

At  the  apex  of  each  synei'gida  a  strongly-refractive, 
homogeneous  cap,  clearly  defined  against  the  fine-grained 
posterior  part,  may  be  easily  seen.  It  is  called  the  fili- 
form apparatus.  Chloriodide  of  zinc  shows  by  the  violet 
reaction  that  this  cap  consists  of  cellulose.  The  rest  of 
the  substance  of  these  cells  and  of  the  ovum  is  colored 


330 


FERTILIZATION    OF    TORENIA. 


yellow-brown  by  this  reagent.  By  careful  examination, 
we  find  that  the  embryo  sac  membrane  is  opened  over  the 
synergidi^  cap,  B,  O.  The  cap,  therefore,  forms  the 
closing  apparatns  for  this  opening  of  the  sac  membrane. 
It  may  be  remarked  in  passing  that  they  are  widely  dis- 


FiG.  lOS.  Torenia  asiatica.  A,  two  ovules  on  the  placentae;  e,  the  free  apex 
of  the  embryo  sac;  e*,  the  lower  widened  part  of  the  same  in  the  interior  of  the 
ovule;  /,  funiculus;  i,  integument.  X  2W.  B  and  C,  free  apices  of  embryo  sac  be- 
fore fertilization;  fl,  synergidse  caps,  filament  apparatus;  o,  egg;  D  and  E,  during 
fertilization;  D,  with  a  part  of  the  funiculus,  /;  t,  pollen  tube.  J5  to  £.  X  600. 

tributed,  particnlarly  in  monocotyledonous  plants  and  are 
often  found  in  them  protruding  some  distance  out  of  the 
embryo  sac.  Their  striation,  which  is  often  observed  in 
these  plants,  is  found  to  consist  of  fine  pores  filled  with 
plasmic  contents. 

By  turning  back  to  our  preparation  we  find  the  distri- 


FERTILIZATION   OF   TORENIA.  331 

bution  of  the  contents  in  the  syncrgiclaä  and  the  ovnm  is 
the  same  as  in  the  Monotropa  and  Orchids,  B,  C.  In 
the  synergidaä  the  nucleus  lies  in  the  upper  part  and  the 
vacuole  in  the  under.     This  is  reversed  in  the  e<r£r. 

If  we  wish  to  study  the  process  of  fertilization  in  the 
Torenia  we  must  pollinate  the  flower.  Thirty-six  hours 
are  required  to  complete  the  process,  so  we  must  begin 
our  examination  a  day  and  a  half  or  two  days  later.  Re- 
move the  ovule  from  the  phicenta  under  the  simplex  Avitli 
the  greatest  possible  care,  taking  therewith  as  much  as 
possible  of  the  pollen  tube.  It  will  then  be  very  easy  to 
trace  its  course  to  the  apex  of  the  embryo  sac  and  down 
between  the  synergida^  caps  to  the  egg,  D,  E.  One  sees 
that  the  pollen  tube  from  the  placenta  leads  down  the  fu- 
niculus till  it  reaches  the  apex  of  the  embryo  sac.  The 
latter  exercises  a  direct  influence  upon  the  direction  of 
the  growth  of  the  pollen  tube.  For  it  is  supposed  that 
the  synergidi\3  secrete  a  substance  which  acts  as  a  kind  of 
stimulus  on  the  pollen  tubes.  On  account  of  the  soft  na- 
ture of  the  caps  they  ofler  little  resistance  to  the  discharge 
of  this  substance.  When  they  are  strongly  developed  they 
are  found  to  be  perforated  with  tine  canals  by  which  the 
secretion  is  discharged.  The  synergidfe  in  Torenia,  as 
elsewhere,  become  disorganized  after  the  entrance  of  the 
pollen  tube,  and  assume  the  refractive  appearance  already 
referred  to.  This  object  is  not  suitable  to  the  further  and 
concluding  process  of  fertilization. 

Notes. 

(1)  Goebel,  Grundzüge  d.  Syst.,  etc.,  p.  417;Lürssen,  Grundz.  d. 
Bot.  p.  35G;  Med.  Pharm.  Bot.  Bd.  ii,  244;Prautl,  Lehrb.  d.  Bot.,  iv 
Aufl.,  p.   195. 

(2)  Strasburger,  Befr.  u.  Zellth.  pp.  34  u.  35. 

(3)  The  same  work,  p.  55. 

(4)  "         "         "        "  54. 
Co)     "         "         "        "  52. 


LESSON  XXX. 
Structure  of  the  Seeds  op  the  Angiosperms. 

We  shall  now  study  the  structure  of  the  ripe  seeds 
and  give  especial  attention  to  the  germ  which  they  bear. 
Take  the  GapseUa  bursa  pastoris,  a  plant  often  used  for 
embryological  studies  (1).  Its  seed  is  relatively  quite 
small,  but  all  the  better  on  that  account  for  an  investigation 
into  the  history  of  its  development.  First,  make  a  longi- 
tudinal median  section,  so 
that  we  may  know  how  the 
object  looks  whose  devel- 
opment we  are  to  study. 
This  may  be  done  without 
too  great  difficulty  Avith  a 
fresh  seed  between  the  fin- 
gers, but  may  perhaps  be 
moi-e  successful!}'  accom- 
plished by  holding  it  be- 
tween two  flat  pieces  of 
cork,  or  by  gumming  it  be- 
tween two  pieces  of  soft 
linden  or  poplar  wood  and 
then  cutting  through  the  wood  and  seed  toijether  when  the 
gum  is  dry;  or,  finally,  the  seed  may  be  embedded  in  the 
end  of  an  elder  stem  Avhich  has  been  hollowed  out,  in  a 
drop  of  gum,  and  when  the  gum  has  dried  cut  the  desired 
section. 

The  section  should  be  examined  in  glycerine,  since  wa- 
ter swells  the  germ  and  so  pushes  it  out  of  the  seed  coat. 
The  germ.  Fig.  109,  A,  fills  the  whole  body  of  the  seed. 

(332) 


Fig.  10!).  ^.longitudinal  section  of  ripe 
seed  of  Capsella  bursa  pastoris;  h,  hypo- 
cotyledon  ;  c,  cotyledon  ;  v.  vascular  bun- 
dle of  funiculus,  X  26.  B,  longitudinal 
section  of  tlie  seed  coat  after  soaking  in 
water;  e,  swollen  epidermis;  c,  brown 
much  thickened  layer;  *,  compressed  lay- 
er; a,  aleuron  layer.  X  210. 


THE    SEED    OF   THE    SHEPHERD's   PURSE.  333 

It  is  bent  double  in  the  miildle,  so  tbut  the  cotyledon,  c, 
lies  next  to  the  hypocotyledon,  li.  This  manner  of  placing 
the  embryo  is  characteristic  of  the  suborder  JSfotorJiizem 
among  the  Cruciferem  and  may  be  represented  by  the 
figure  110.  If  the  section  is  strictly  median  and  suffi- 
ciently delicate  as  in  the  figure,  one  may  see  the  small 
vegetative  cone  of  the  stem  at  the  base  between  the  ^coty- 
ledons, and  also  at  the  radicular  end  of  the  hypocotyle- 
don a  root-cap  but  a  few  cell  layers  thick.  There  is  no 
endosperm,  but  the  germ  is  surrounded  immediately  by 
the  seed-shell  or  testa.  If  we  now  take  a  stronger  mag- 
nification  we  shall  be  able  to  demonstrate  that  this  seed 
coat  is  composed  of  three  layers  of  cells,  Fig.  109,^,  an 
inner  layer  of  cells,  «,  of  relatively  thin,  colorless  walls 
and  granular  contents.  Testing  by  a  solution  of  iodine, 
•we  find  the  grains  colored  a  yellow-brown  and  therefore 
must  be  aleuron  or  proteid  mattei".  Next  to  this  layer  is 
one,  c,  w'ith  the  cell  walls  a  deep  brown  and  nmch  thick- 
ened towards  the  inside.  The  outer  cell  layer  appears 
like  a  colorless,  homogeneous  membrane  in  concentrated 
glycerine,  its  cells  being  much  flattened  and  thickened 
till  the  cell  cavity  quite  disappears ;  between  the  inner 
and  second  layer  of  cells  is  a  layer  of  flat,  compressed  cells 
which  appears  to  be  a  simple  membrane.  If  we  look  at 
the  seed  from  the  outside  we  shall  easily  recognize  the 
outline  of  the  polygonal  cells  wdiich  make  up  the  outer 
layer.  These  cells  are  separated  in  part  in  their  inner 
portion  by  air-filled,  intercellular  spaces.  In  the  middle 
of  each  cell  is  a  part  which,  not  distinctly  marked  in  it- 
self, is  strongly  refractive.  The  walls  of  the  next  inner 
layer  are  brown,  much  thickened,  the  cells  themselves 
only  a  little  smaller  than  those  of  the  outer  layer.  Con- 
siderably smaller  and  much  less  thickened  are  the  cells  of 
the  third  layer  which  contains  the  gluten  grains. 


334         STRUCTURE  OF  THE  SEED  COAT. 

'  If  we  now  admit  a  little  water  from  the  edge  of  the 
cover-glass,  the  cells  of  the  outer  layer  will  rapid  1}^  swell 
and  put  out  a  strongly  refractive  little  column  from  the 
middle  of  each.  The  whole  cell  cavity  disappears  being 
filled  with  the  thickening  layer  of  the  wall.  The  inner- 
most thickening  layer  forms  the  remarkable  column  which 
protrudes  from  the  surface.  The  intercellular  spaces  have 
disappeared.  The  swelling  walls  are  distinctly  laminated. 
B}^  further  addition  of  water,  the  cuticle  of  the  cell  will 
be  detached  and  the  outer  thickening  layer  will  dissolve 
in  the  water  in  invisible  mucilage.  The  column  remains 
indicating  the  position  of  the  middle  of  each  cell,  Fig. 
109,  B,  e.  It  has  not  inconsiderably  increased  in  size 
and  one  may  see  the  remainder  of  the  dissolved  thicken- 
ing layer  attached  to  its  apex.  The  middle  lamella  Avhich 
remains,  not  having  swollen,  is  not  so  high  as  the  column. 
All  this  is  seen  in  the  illustration.  Fig.  109,  which  rep- 
resents a  section  of  the  testa  after  it  has  been  subjected 
to  the  action  of  water.  All  this  can  be  seen  taking  place 
more  rapidly  if  the  section  be  first  examined  in  alcohol 
and  then  water  added. 

The  thickening  layer  of  the  outer  cells  of  many  seeds 
shows  this  peculiarity  of  forming  a  mucilage  in  water.  It 
serves  the  double  purpose  often  of  gluing  the  seed  to 
foreign  objects  which  carry  it  far  from  the  parent  plant, 
and  also  furnishes  a  viscous  reservoir  of  water  upon  the 
outside  of  the  seed. 

Since  it  is  dijfficult  to  make  sections  of  perfectly  ripe 
seeds  it  is  better  when  we  only  wish  to  study  the  position 
and  the  structure  of  the  embryo,  to  take  seeds  which 
are  yet  öoft  and  unripe,  and  use  the  wholly  ripened  seeds 
only  in  a  study  of  the  testa.  We  will  now  go  back  to 
the  younger  stage  and  put  the  whole  embryo  seed  in  pot- 
ash lye.     We  shall  get  these  unripe  seeds  best  by  splitting 


DEVELOPMENT   OF   THE    SEED.  335 

the  little  pod  in  halves  and  then  with  a  scalpel  taking  them 
out  of  the  halves.  Till  the  seed  is  almost  ripe  it  may  be 
thus  made  sufficiently  transparent  to  enable  us  to  see  the 
exact  position  of  the  embryo.  The  embryo  becomes  a 
beautiful  green  in  the  potash  which  shows  that  the  starch 
grains  swell  and  the  chlorophyll  grains  become  thereby 
visible.  AVe  see  thereby  that  the  embryo  is  shorter  the 
younger  the  seed  we  examine,  and  especially  is  this  true 
of  the  cotyledons.  It  is  withdrawn  more  and  more  from 
the  under  half  of  the  cavity  of  the  embryo  sac  which  is 
bent  upwards.  Seed  buds  from  pods  which  without  the 
style  measure  not  more  than  half  a  centimeter  in  length 
show  the  emlnyo  as  a  small  heart-shaped  body.  The  two 
projecting  separating  processes  are  the  beginnings  of  the 
cotyledons. 

As  we  follow  the  successive  stages  of  the  development 
of  the  seed  w^e  shall  see  that  the  endosperm  is  formed  only 
at  the  two  ends  of  the  embryo  sac  and  principally  at  the 
chalaza  end  as  a  green  tissue  body.  Only  in  the  nearly 
ripened  seed  will  this  be  reached  and  supplanted  by  the 
cotyledons.  We  also  shall  see  that  the  testa  is  formed, 
the  outer  layer  from  the  outer  integument  of  the  ovule, 
and  the  inner  layer  of  cells  from  the  inner  integument. 
The  latter  is  earlj^  distinguished  by  its  rich  cell  contents. 
Between  the  inner  and  outer  integument  is  a  layer  of  cells 
one  or  two  cells  thick  which  gradually  is  extended  and 
compressed  till  it  finally  forms  a  thin  membrane  between 
the  second  and  third  layers  of  the  seed  coat. 

In  order  to  study  the  egg-apparatus  of  the  ovule  at  the 
time  of  its  fertilization,  we  must  use  alcohol  material  which 
we  have  previously  sufficiently  clarified  with  potash  lye. 
We  seethe  two  synergidfeand  the  oospore  in  the  egg  appa- 
ratus but  the  antipodal  cells  are  very  difficult  to  make  out. 
The  structure  of  the  ovule  is  easily  traced  in  the  fresh 


336  DEVELOPMENT   OF    THE    GERM. 

material  examined  in  water,  or  when  made  more  trans- 
parent by  the  addition  of  a  little  potash.  The  ovnle  is 
campylojtropic  ;  that  is,  its  nncellus  and  embryo  sac  are 
bent  double  as  we  have  ah'eady  seen  in  the  older  seeds. 
The  outer  integument  consists  of  two  layers  of  cells,  the 
inner  in  its  upper  part  of  two  and  further  along  of  three. 
In  this  stage  of  development  the  nuoelkis  is  alread}^  sup- 
planted, so  that  the  embryo  sac  rests  directly  upon  the  in- 
ner integument.  The  funicuhis  is  pretty  long  and  is 
furnished  with  a  vascular  bundle  which  hoAvever  ends  at 
the  chalaza  and  is  still  to  be  seen  in  the  ripe  seed,  Fig. 
109,  A,  V.  The  next  stage  in  the  development  is  very 
interesting.  It  may  be  studied  without  the  aid  of  potash 
lye.  We  observe  that  the  fertilized  oospore  has  grown 
out  into  a  filamentous  protogerm  about  six  cells  long.  The 
upper  part  of  this,  that  is  the  cell  farthest  removed  from 
the  micropyle,  rounds  out  into  an  embryosphere,  while  the 
lowermost  cells  of  the  embryo-carriers,  or  suspensors,  puff 
up  bladder-like,  supplant  the  whole  nucellus  tissue  quite 
to  the  integument  and  form  the  bladder  Avhich  we  find  in 
this  place  already  complete. 

In  such  preparations  we  are  al)le  to  see  that  the  embryo 
spherule  is  separated  from  the  suspensor  b}"  a  division 
wall,  and  is  then  parted  longitudinally  by  an  hoi'izontal  wall 
and  again  by  another  like  wall  at  right  angles  to  the  first 
and  then  finall}^  into  eight  parts  by  a  transverse  division 
wail.  The  embryo  spherule  increases  in  size  and,  in  the 
number  of  its  cells,  becomes  a  little  compressed  at  its  ante- 
rior end  from  which  the  cotyledons  grow.  Between  the 
bases  of  these  is  the  veo-etative  cone  of  the  stem. 

For  a  study  of  the  germ  of  the  monocotyledons,  we  will 
take  the  common  water  plantain  Alisma  plantago  (2). 
First  of  all,  we  will  make  ourselves  familiar  with  the  full 
o-rown  form.     The  blossom  contains  several  monomerous 


SEED    OF    ALISMA. 


ovaries.  It  is  poly  carpal.  From  each  blossom  several 
seeds  are  produced  pressed  closely  together  forming  a 
compound  fruit  of  triangular  outline.  Each  seed  is  much 
compressed,  thicker  above,  a  reverse  ovate  in  form  with  a 
median  furrow  on  the  back.  On  the  inside  edge  of  each 
seed  about  iialf-way  up  projects  a  short  filiform  process 
corresponding  to  the  withered  pistil.     For  our  section  we 

will  take  a  seed  not 
quite  ripe  and  be- 
tween the  two  halves 
of  a  cork  stopper 
make  oiu*  section,  the 
seed  coat  being  too 
haid  to  d(j  it  conven- 
iently while  holding- 
it  with  the  fino;ers. 
We  will  also  make 
some  transections  in 
the  same  way.  The 
longitudinal  sections 
„„     ^,.        ,    .        ,,  ,•     ,      ^  ,•      should   be    examined 

Fig.  110.    Ahxma  plantago.    Median  longitudi- 
nal section  of  lipe  fruit;  e^j,  epical)»  (eiiideiiuis^ ;  in     water     to      which 
»H,  mesocarp;  en,  endocavp  of  tlie  fruit  wall,  peri-  f      1      1  i 
carp;  t>,  vascular  bundle;  «;*,  end  of  same;   st,  SOUIÄ    potasll    lye    lias 
dead  pistil;  t,  style;  /,  funiculus  of  seed  witli  beCIl  added.      For  the 
vascular  bundle, /r;  »rep,  micro pyle;  cä,  clialaza 

end;  <s,  seed  coat,  testa;  Ap  hypocotyledou; /7,  transections  purC  Wa- 

primary  leaf;  d,  cotyledon.  X  ■i«-  j-g^.  ^yill  do. 

To  remove  the  air  from  the  former  section  for  the  ex- 
amination of  the  seed  coat  it  nniy  be  put  for  a  short  time 
in  alcohol  or  under  the  air  pump.  Lay  some  of  these 
secti(ms  also  in  carbolic  acid  and  so  obtain  views  of  the 
structure  which,  in  important  respects,  supplements  those 
obtained  from  the  other.  The  lonoitudinal  section  is  il- 
iustrated  in  Fig.  110.  AVe  have  first  the  relatively  thick 
pericarp  covered  with  the  epidermis,  ep.      The  latter  is  a 


338  SEED    OF   ALISMA. 

pretty  clearly  distinct  part  of  the  pericarp  and  may  be 
called  the  epicarp.  Next  to  this  is  the  mesocarp,  m,  pa- 
renchymatous tissue  of  very  nearly  isodiametric  cells  filled 
with  air.  The  endocarp,  e?i,  consists  of  several  layers  of 
elongated  sclerenchyma  elements.  An  exactly  median 
section  will  open  a  mucilage  passage  on  the  back  side  of 
the  pericarp.  This  can  be  best  seen  in  the  unripe  seed, 
since  in  the  ripe  it  is  almost  empty  of  contents  and  is 
scarcely  distinguishable  from  the  surrounding  tissue.  A 
section,  not  exactly  median,  will  lay  bare  a  vascular 
bundle,  resting  on  the  endocarp,  and,  entering  on  the  back 
side,  V,  passiug  over  and  down  to  the  front  of  the  fruit  to 
V* ,  At  the  point  where  the  pistil,  s^,  is  iu'^erted,  the 
pericarp  projects  in  a  sharp  edge  formed  of  elongated 
cells.  \\\  favorable  cases,  an  air-tilled  passage,  t,  endiug 
at  the  pistil  and  extending  nearly  to  the  seed  cavity,  may 
be  made  out.  It  is  the  way  by  which  the  pollen  tube 
reaches  the  ovule.  Since  the  ovule  has  its  micropyle 
turned  towards  the  back  side  of  the  ovary,  the  pollen  tube 
must  pass  around  the  funiculus  after  entering  the  cavity 
of  the  ovary. 

The  layers  of  the  pericarp  and  the  furrow  in  the  back 
side  of  the  fruit  are  more  distinctly  seen  in  the  transection, 
than  in  the  longitudinal  section.  As  is  seen  in  the  latter 
section  the  seed  very  nearly  fills  the  cavity  of  the  ovary 
and  is  held  in  a  central  position  at  the  base  of  this  cavity 
by  a  pretty  long,  bent  funiculus, /i  The  vascular  bundle 
enters  by  the  funiculus.  The  seed  is  campylotropic  and 
is  completely  filled  by  the  embryo.  The  testa,  ^s,  is  a  thin 
membrane  consisting  of  two  distinct  cell  layers,  but  a  third' 
may  be  sometimes  seen  after  treatment  with  potash  lye. 
The  micropyle  projects  shaiply  from  the  seed.  The  root 
end  of  the  germ  lies  directly  within  the  micropyle.  On 
the  left  and  within  about  half-way  up  the  seed  is  a  small 


STRUCTURE    OF    ALISMA    SEED.  339 

indentation  in  the  embryo.  Here  lies  the  vegetative  cone 
of  the  stem  and  from  it  arises  the  beginning  of  the  first 
leaf,/ 1,  which  completely  fills  the  little  cavity.  The  hypo- 
cotyledon  lies  between  the  vegetative  cone  and  the  root, 
is  covered  with  an  epidermis,  and  has  three  layers  of  rind 
cells  regularly  arranged,  and  a  central  cord  of  elongated 
cells  which  extends  from  the  apex  of  the  root  towards  the 
vegetative  cone.  These  rind  layers  have  but  one  common 
initial  layer  at  the  apex.  The  dermatogen  runs  over  this 
from  which  the  two  root-caps  seem  to  be  derived.  The 
hypocotyledon  continues  into  the  cotyledon  which  may  be 
seen  in  the  germinal  cavity  bent  double,  gradually  dimin- 
ishing in  size  towards  the  end,  which  terminates  at  the 
chalaza  end  of  the  seed.  The  cotyledon  also  consists  of 
concentrically-arranged  layers  of  cells  wrapped  around  a 
central  cord  of  elongated  cells.  This  cord  bends  under  the 
vegetative  cone  and  continues  into  the  hypocotyledon. 
The  cell  layers  of  the  rind  also  pass  from  the  latter  over 
into  the  cot3'ledon.  The  cell  layers  of  the  rind  diminish 
upwards  towards  the  attenuated  point  of  the  cotyledon 
from  three  to  one,  the  central  cord  ending  some  distance 
below  the  apex  of  the  cotyledon.  There  is  no  trace  of  an 
endosperm  in  the  ripe  seed.  The  embryo  is  closely 
packed  with  starch  in  all  its  cells. 

A  transection  sives  nothin«:  new,  but  shows  the  con- 
centric  arrangement  of  the  cells  very  clearly,  and  aftbrds 
a  better  view  of  the  structure  of  the  testa  than  the  lon^i- 
tudinal  section. 

These  two  illustrations  of  the  method  of  formino:  the  em- 
bryo  in  the  angiosperms  are  typical  forms  of  the  dicotyle- 
dons and  monocotyledons,  but  are  very  far  from  being 
typical  of  all  the  cases  which  have  been  observed.  For 
there   are  dicotyledons  which  possess  but  one   germinal 


340  LITERATURE    OF   LESSON. 

leaf  {Varum  bulbocastanmn,  Ranunculus  ficaria')^  and 
monocotyledons  where  tlie  germinal  leaf  is  produced  lat- 
erally from  the  adjacent  terminal  vegetative  cone  of  the 
stem,  Dioscoracece,   Oomtnelyneoe  (3). 

Notes. 

(1)  See  Hansteiu,  Bot.  Abliandl.  Bd.  i,  Heft  1,  p.  5;  Westermaier, 
Flora  187G,  p.  483;  Fammtziu,  Mem.  de  I'Acad.  imp.  d.  sc.  d.  St.  Pe- 
tersb.,  VII  ser.,  T.  xxvi,  N.  10;  Kny,  bot.  Wandtafeln,  Heft  i,  p.  20. 
Eine  Zusammenstellung  aller  enibryologischen  Arbeiten  in  Goebel, 
Vergl.  Entwicklungsgeschichte,  in  Schenk's  Handb.  d.  Bot.  Bd.  iii, 
p.  165,  ff. 

(2)  Hanstein,  quoted  above,  p.  33;  Famintzin,  quoted  above,  p.  4. 

(3)  The  literature  in  Goebel,  1.  c.  p.  169,  ff. 


LESSON  XXXI. 

The  Fkuit  of  the  Angiosperms. 

For  the  study  of  a  fruit  capsule  of  more  complicated 
structure  thau  that  of  the  orchid  already  examined  Ave  will 
take  a  ripe  plum,  Prunus  domestica.  On  the  surface  is  a 
delicate  covering  of  down.  The  epidermis  of  the  plum 
is  composed  of  cells  arranged  in  groups  which  betray 
their  origin  in  a  common  mother-cell.  They  contain  a 
rose-red  cell-sap.  A  delicate  transection  will  show  that 
the  cells  under  the  epidermis  for  several  laj^ers  deep  rap- 
idly increase  in  size  and  then  remain  of  like  size  beyond. 
They  are  rounded  up  towards  each  other  and  yet  form 
only  small  intercellular  spaces.  They  contain  a  few  very 
small  yellowish-green  chlorophyll  grains,  a  thin  wall  layer 
of  protoplasm,  and  a  nucleus,  elsewhere  colorless  cell-sap. 
This  tissue  which  is  penetrated  with  numerous  vascular 
bundles  is  composed,  nearer  the  stone,  of  smaller  and  ra- 
dially elongated  cells.  The  stone  itself  cannot  be  cut  with 
a  razor  without  danger  of  breaking  the  blade.  If  such 
an  instrument  is  to  be  used  a  surface  must  be  careful!}'  pre- 
pared for  cutting,  with  a  pocket  knife.  The  cell  walls  will 
be  found  to  be  much  thickened  and  lignified  and  penetrated 
with  delicate,  branched  canals.  A  study  of  the  develop- 
ment of  the  fruit  will  show  that  the  whole  of  the  tissue  of 
the  plum,  including  the  stone,  takes  its  origin  from  the  walls 
of  the  ovary,  the  epidermis  of  the  plum  from  that  of  the 
ovary,  the  pulp  from  the  mesocarp,  the  stone  from  the  in- 
ner tissue,  the  endocarp.  Within  the  stone  is  the  seed 
which  consists  of  the  germ,  the  delicate  seed  membrane 

(3^1) 


342        STRUCTURE  OF  PLUM  AND  APPLE. 

and  the  endosperm.  A  transection  shows  us  the  two  flat 
cotyledons,  and  a  median  longitudinal  section  will  show 
at  the  base  between  the  cotyledons  the  stem  of  the  germ 
with  its  root  end  at  the  pointed  micropyle  end  of  the  seed, 
and  the  plumule  between  the  cotyledons  at  their  base. 
The  embryo  finally  supplants  the  whole  original  seed  tis- 
sue quite  to  the  thin  testa,  on  which  laterally  from  the  mi- 
cropyle the  funiculus  projects.  A  delicate  transection 
shows  the  testa  to  be  composed  of  a  compressed  cell-layer 
beset  without  by  a  few  or  several  rounded  cells.  Between 
the  testa  and  the  cotyledons  is  an  endosperm  either  wholly 
suppressed  or  reduced  to  a  layer  of  cells.  The  rounded 
cells  scattered  over  the  surfjice  of  the  testa  are  epidermal 
cells  of  the  testa  which  have  become  thickened  while  their 
neighbors  have  remained  unthickened  and  have  been  com- 
pressed. The  testa  arises  from  the  one  integument  of  the 
ovule.  Two  ovules  occur  in  the  ovarj^  but  one  only  is  de- 
veloped. 

The  observations  made  upon  the  plum  will  serve 
equally  well  for  the  cherry  with  unimportant  differences. 

We  will  next  examine  the  structure  of  the  apple.  While 
the  plum  and  cherry  form  their  superior  ovary  from  a  sin- 
gle carpel,  the  inferior  five-celled  ovary  of  the  apple  is 
formed  from  a  union  of  five  carpels.  As  in  the  nearly  re- 
lated form,  the  rose,  the  five-celled  ovary  may  be  supposed 
to  be  an  excavated  stem,  a  so-called  hypanthium.  To  des- 
ignate the  apple  as  well  as  the  hawthorn-l)erry  a  pseudo 
fruit  is  in  all  respects  incorrect,  since  it  differs  in  noth- 
ing from  the  inferior  ovaries  of  many  other  plants. 
The  apple  is  crowned  at  its  apex  with  five  more  or  less 
perfectly  dead  sepals  and  the  dried  up  parts  of  the  flower. 
A  superficial  section  shows  that  the  epidermis  of  the  ap- 
ple is  formed  of  relatively  small  polygonal  cells  by  the 
arrangement  of  which  it  is  possible  to  follow  the  successive 


STRUCTURE    OF   APPLE.  343 

steps  of  their  development.  The  walls  of  the  cells  are 
considerably  thickened,  their  cell-sap  either  colorless  or 
rose-red.  The  surface  is  covered  with  a  finely  granuLu* 
waxy  substance.  The  minute  elevations  of  the  surfaces 
which  are  easilv  seen  with  the  mao-nifyino^  ojlass  are  occu- 
pied  in  the  middle  with  a  stoma.  The  tissue  under  these 
stomata  is  often  dead ;  and,  the  surface  cracking,  the 
wound  is  finally  closed  with  a  growth  of  cork.  A  thin 
transection  will  show  us  that  the  epidermis  is  much  thick- 
ened on  the  outside  and  that  below  this  the  cells  for  sev- 
eral layers  deep  are  radially  elongated,  with  thickened 
walls,  which  gradually  become  larger  and  thinner  walled 
within,  and  contain  chlorophyll.  There  is  therefore  no 
sharp  demarcation  between  epicarp  and  mesocarp.  The 
chlorophyll  grains  are  closely  packed  with  starch.  Their 
color  disappears  and  they  diminish  in  number  towards  the 
interior  of  the  apple.  At  a  certain  depth  the  large,  blad- 
der-like cells  of  the  mesocarp  have  only  a  wall  lining  of 
lu'otoplasm  and  a  nucleus,  besides  the  cell-sap,  the  inter- 
cellular spaces  being  tilled  with  air. 

The  five  seed  chambers  are  covered  with  a  smooth, 
hard  membrane,  the  endocarp,  which  corresponds  to  the 
stone  of  the  plum.  It  consists  of  several  layo's  of  scle- 
renchyma  fibres  which  are  irregularly  bevelled,  often 
bent  and  run  into  dilferent  layers.  The  five  compart- 
ments often  separate  from  each  other  in  the  middle, 
forming  a  central  cavity  into  which  they  most  generally 
open.  At  the  bottom  of  each  chamber  are  two  ovules, 
one  or  both  or  generally  neither  of  which  develop  into 
seeds.  The  seed  is  nearly  filled  with  the  germ  which 
has  the  same  structure  as  that  of  the  plum  or  cherry,  the 
testa  being  much  thicker.  A  transection  of  the  epider- 
mis shows  it  to  consist  of  an  outer  layer  of  colorless  and  an 
inner  layer  of  brown  cells,  the  former  being  capable  of 


344  STRUCTURE  OF  APPLE  SEED. 

much  swelling,  the  Litter  not.  If  a  section  be  put  in  water, 
the  former  l;iyer  soon  greatly  increases  in  thickness,  and 
the  cells  of  the  cuticle  expand  and  arch  out  papillately. 
This  is  Avhat  makes  the  moist  seed  slippery.  The  tissue 
immediately  beneath  this  consists  of  polygonal  cells 
rounded  at  the  corners,  much  thickened  and  brown.  Be- 
low this,  follows  a  layer  about  a  third  as  thick,  formed  of 
tangentially-elongated,  brown,  somewhat  thinner  cells. 
These  border  on  the  blight,  white,  thick  membrane. 
The  latter  arises  from  the  strongly-thickened,  outer  wall 
of  the  centre  nucellus  layer,  the  whole  of  the  rest  of  the 
testa  from  the  outer  integument  of  the  ovule.  The  inner 
integument  is  suppressed  earlier.  The  nncollar  cells 
"which  we  have  reckoned  in  the  testa  are  mostly  com- 
pressed, as  are  also  the  remaining  cells  of  the  nucellus. 
On  these  flattened  cells  follows  a  thin  layer  of  endosperm 
which  is  in  some  places  quite  supplanted,  but  elsewhere 
covers  the  embryo.  The  endosperm  cells  are  closely 
packed  with  gluten  grains.  Successive  superficial  cells 
show  that  the  epidermis  consists  of  relatively  few  elon- 
gated cells  whose  inner  thickening  layer  is  porous.  The 
next  following  tissue  consists  of  elongated  cells  with  di- 
agonally arranged  dots.  The  tangentially-elongated,  in- 
ner elements  of  the  testa  are  at  right  angles  with  these. 

A  transection  of  a  ripe  orange,  Clirus  vulgaris(l),  shows 
us  compartments  varying  from  six  to  twelve,  laterally  sep- 
arated by  their  partition  Avails  and  filled  with  orange-red 
pulp.  The. partitions  meet  in  a  central  tissue-column. 
We  may  consider  the  outer  shell  or  rind  the  epicarp,  the 
pulp,  the  mesocarp,  the  central  column  and  partition  walls 
the  endocar}).  A  delicate  transection  of  the  outer  rind 
shoAvs  a  small-celled  epidermis  followed  by  tissue  of  grad- 
ually larger  cells.  The  epidermis  and  the  next  folloAving 
tissue  have  orange-yellow  chromatophores.     Then  inter- 


STRUCTURE    OF    ORANGE.  345 

cellular  spaces  filled  with  air  follow  which  gradually  become 
so  large  as  to  give  the  tissue  the  character  of  loose  sponge- 
parenchyma.  The  cells  are  tangentially  elongated.  The 
shell  is  permeated  with  vascular  bundles  which  the  tran- 
section usually  lays  bare  lengthwise  and  which  branch  out- 
ward towards  the  surface.  In  the  epidermis  we  see  with 
the  naked  eye  the  large  receptacles  of  essential  oil,  which 
are  of  the  same  structure  as  those  of  Ruta  and  are  lined 
with  a  layer  of  delicate  cells.  These  oil-cavities  appear 
like  dark  spots  in  the  fruit.  A  superficial  section  shows  the 
cells  overlying  these  receptacles  to  be  lacking  in  the 
orange-red  chromatophores  and  to  contain  in  their  place 
large  gloI)ales.  A  deeper  cut  shows  the  structure  of  the 
various  oil-holders,  and  the  vascular  bundles  between 
them.  Still  deeper  sections  bring  us  to  the  sponge-like 
tissue  formed  from  cells  elongated  into  tubes.  In  the  tis- 
sue next  to  the  semnents  the  cells  of  the  rind  are  lonoer, 
fil)rous,  and  in  part  more  thickened  and  furnished  with 
diagonally  placed  pits.  So  also  the  partitions  between  the 
segments  are  built  of  sponge-like  elements  on  the  inside 
and  of  elongated  thickened  cells  without.  The  former 
easily  separate  from  their  connection,  the  latter  adhere 
pretty  well  together. 

In  separating  the  segments  in  the  usual  way  the  sponge 
tissue  parts  while  the  tibrous  tissue  remains  as  a  soft  white 
covering  to  the  pulp.  This  membrane  may  be  used  di- 
rectly for  examination.  It  will  be  found  to  consist  of  un- 
thickened  and  thickened  fibrous  cells,  the  latter  pitted. 
The  pulp  consists  of  club-shaped  tubes  which  arise  on  the 
outside  of  the  compartment.  They  are  inserted  with  a 
slender  basis,  and  pressed  together  fill  the  segment.  The 
larger  they  are  and  the  deeper  they  extend  into  the  seg- 
ment the  more  do  they  take  on  a  radial  arrangement  at 
right  angles  to  the  longer  axis  of  the  segment.     Each  of 


346         DEVELOPMENT  OF  THE  ORANGE. 

these  club-shaped  elements  shows  that  it  is  surroiincled  by 
a  layer  of  connected  elongated  fihn-like  cells  similar  to 
those  which  we  found  in  the  partition  wall  between  the 
segments.  They  are  much  thickened  and  furnished  with 
diagonally-placed  pits.  The  inside  of  these  club-shaped 
tubes  is  filled  with  large,  polygonal,  thin-walled  cells  filled 
with  sap,  in  whose  interior  are  visible  slender,  spindle- 
shaped,  orange-red  chromatophores.  The  central  column 
where  it  joins  the  partition  walls  consists  of  the  same 
sponge-parenchyma  which  forms  the  inner  part  of  the 
"  peel  "  of  the  orange.  In  the  pulp  is  embedded  an  in- 
definite number  of  seeds.  They  occupy  the  inner  edge  of 
the  segment,  their  place  of  insertion  being  turned  inward. 
By  removing  the  segment  the  seed  is  separated  from  the 
placenta.  In  most  cases  a  part  of  the  tissue  of  the  central 
column  together  with  the  placenta  remains  adhering  to 
the  iimer  edge  of  the  segment. 

We  will  make  a  study  of  the  development  of  the  fruit 
of  the  orange  tree,  with  reference  only,  however,  to 
learning  the  more  important  phases  of  its  history.  A 
transection  of  an  ovary  which  has  but  just  shed  its  blos- 
som shows  already  a  pretty  thick  envelope,  while  the 
segments  are  relatively  small,  the  central  column  stout 
and  the  oil  receptacles  already  developed  in  the  rind. 
The  ovules  are  inserted  in  two  rows  in  the  inner  ano-le  of 
the  segments  with  their  lonsrer  axis  extended  outward. 
The  s.egments  are  enveloped  with  an  epidermis,  on  which 
border  two  or  three  other  closely  compacted  layers  of 
tissue,  while  further  in  the  tissue  contains  air-filled 
spaces.  From  the  outer  surface  of  each  segment  already 
small  knobs  project  inwardly.  The  inner  epidermis  and 
the  next  following  cell  layers  partake  in  the  formation  of 
these  knobs.  A  transection  of  a  small  fruit  not  more 
than  5  mm.  in  diameter,  will  show  us  in  place  of  these 


DEVELOPMENT  OP  THE  ORANGE.         347 

knobs,  cylindrical,  small-celled  protuberances,  which 
reach  different  depths  in  the  segment  and  begin  to  pen- 
etrate between  the  embryo  seeds.  Their  epidermis  con- 
tinues into  that  of  the  segments,  while  their  inner  cells 
pass  over  into  the  hypodermal  tissue  which  surrounds  the 
segments.  These  protuberances  are  still  found  in  earlier 
stages  of  the  development  of  the  fruit,  the  cells  of  their 
surface  being  papillaceous.  The  older  the  young  fruit 
is  which  we  examine  the  longer  will  be  the  tubes  which 
form  the  pulp  of  the  segments.  The  segments  them- 
selves remain  still  very  small  in  comparison  with  the 
thickness  of  the  growing  rind,  in  the  periphery  of  which 
are  the  increasing  oil  receptacles.  The  pulp  tubes  begin 
further  on  to  assume  a  club  shape  in  their  upper  part  and 
to  cover  themselves  Avith  epidermis  lengthwise,  while 
their  inner  cells  remain  isodiametric  by  repeated  trans- 
verse divisions.  These  cells  are  tilled  with  yellowish  con- 
tents. A  considerable  expansion  of  the  epidermis  which 
covers  the  segments  takes  place  and  also  of  the  layers 
which  lie  next  to  that  and  which  were  previously  dis- 
tinguished by  lack  of  intercellular  spaces.  This  all 
takes  place  in  a  young  orange  not  over  15  or  20  mm.  in 
diameter  and  essentially  exjj^ains  the  method  of  develop- 
ment, for  the  pulp  tubes  are  not  further  differentiated 
than  is  needful  to  attain  the  condition  seen  in  the  ripe 
fruit.  But  from  the  epidermis  of  the  segments  there 
arises  the  fibrous  layer  which  covers  the  pulp  tulies.  The 
loose  tissue  of  the  central  column  and  that  of  the  princi- 
pal rind  furnish  the  sponge  parenchyma.  In  the  periph- 
ery of  the  rind  the  oil  cavities  may  be  found  in  every 
stage  of  development,  and  the  layers  which  now^  contain 
chlorophyll  are  those  which  at  a  later  period  contain  the 
orange-red  chromatophores. 

A  transection  of  an  ovary  in  blossom  treated  with  potash 


348      DEVELOPMENT  OF  OVULE  OF  ORANGE. 

shows  US  easily  an  ovule  in  median  longitudinal  section(2). 
The  ovules  are  anatropic.  We  easily  make  out  two  thick  in- 
teguments, a  nucellus,  and  in  an  exactly  median  section  a 
small  embryosac.  Four  weeks  will  elapse  between  the 
pollination  and  fertilization  of  the  orange.  There  is  diffi- 
culty in  studying  the  fertilizing  process,  but  if  we  take  an 
ovule  from  a  young  fruit  about  20  mm.  thick  we  can  easily 
make  a  longitudinal  section  between  the  thumb  and  fino;er 
and  lind  in  the  apex  of  the  embryo  sac  the  minute  germ. 
The  nucellus  is  excavated  and  the  course  the  pollen  tubes 
take  to  come  to  the  embryo  sac  is  marked  by  small  cells  rich 
in  contents.  The  inner  integument  is  recognized  by  the 
brown  color  of  its  inner  cell  layer  and  the  smaller  size  of 
its  elements.  The  outer  integument  is  considerably  thicker 
than  the  inner.  On  the  latter  the  epidermis  begins  to  fill 
itself  with  fine  grained  contents  on  the  inside  and  to  thicken 
on  the  outside.  If  the  ovule  has  reached  a  height  of  3-5mm. 
a  very  peculiar  appearance  is  to  be  seen.  In  the  imme- 
diate neighborhood  of  the  apex  of  the  embryo  sac  a  pro- 
tuberance arises  which  is  visible  in  the  tissue  of  the 
surrounding  nucellus.  It  will  produce  in  this  genus  as  in 
a  number  of  other  angiosperms  an  adventive  germ  along 
with  the  fertile  ovum.  A  median  longitudinal  section 
through  the  next  older  seed-emluyo  will  show  us,  in  dif- 
ferent stages  of  development,  roundish  germ  buds  project- 
ing into  the  embryo  sac  clustered  especially  in  the  anterior 
end.  Soon  the  endosperm  begins  to  develop  and  in  the 
next  older  stage  we  find  the  embryo  sac  quite  filled  with  it. 
In  the  latter  the  embryo  germ  soon  begins  to  develop  the 
cot3dedons  which  assume  the  form  typical  of  the  dicotyle- 
donous plants.  The  nucellus  is  repressed  quite  to  the  out- 
er cell  layer  of  the  eml)ryo  sac.  The  epidermal  cells  on  the 
outer  integument  have  been  considerably  elongated,  and 
much  thickened  without.     The  rest  of  the  tissue  of  the 


SEEDS    OF   THE    ORANGE.  349 

outer  and  inner  integument  has  undergone  no  essential 
chanixe.     The   germs  soon  begin  to    interfere  with  each 

CO  o 

other's  development,  one  or  the  other  getting  the  upper 
hand  after  the  endosperm  is  suppressed,  and  filling  out  the 
embryo  sac.  A  longitudinal  section  of  a  ripe  seed  will  show 
us  one  or  more  germs  pressed  upon  each  other  and  with 
these  perhaps  several  which  have  had  their  development 
arrested.  The  polyembryonic  nature  of  the  orange  seed 
does  not  rest  on  the  existence  in  the  embryo  sac  of  several 
ova  capable  of  fertilization  but  on  the  growth  of  adventive 
germs.  The  testa  is  produced  from  the  two  integuments 
and  the  outer  cell  layer  of  the  nucellus,  which  latter  is  full 
of  rich  cell  contents.  The  boundary  between  the  integu- 
ments has  disappeared,  but  the  inner  layer  of  the  inner  is 
distinguished  by  its  color.  The  epidermis  on  the  outer 
integument  has  attained  a  considerable  height,  and  by 
means  of  a  newly-formed,  diagonally-pitted  thickening 
layer  is  also  much  thickened  on  its  side  walls.  The  outer 
thickening  mass  swells  in  water  making  the  seed  slippery 
and  slimy.  The  inner  thickening  layer  Avhich  is  pro- 
duced last  increases  in  volume  and  forms  papillate  pro- 
jections without. 

Notes. 

(1)  See   also  Poulsen,  Botaniska  Notisei"  utg.  of  Nordstedt  1877, 
p.  97.  There  the  literature. 

(2)  E.    Strasburger,   Jen.    Zeitschr.    f.  Naturw.  Bd.  xir,  1878,    p. 
C52. 


LESSON  XXXII. 
Self-division  of  Nucleus  and  Cell. 

The  best  object  for  studying  the  process  of  cell  and 
nucleus  division  is  the  already  known  hairs  of  Tvadescantia 
virginica  or  of  some  related  species  (1).  We  should 
take  the  hair  before  it  is  fully  grown,  and  when  it  is 
undergoing  a  lively  increase  in  its  cells.  Use  a  bud  5  or 
6  mm.  high.  First  open  the  bud  and  with  a  tine  forceps 
remove  the  anthers  from  the  filaments.  Then  cut  the 
ovary  across  below  the  insertion  of  the  filaments.  Under 
the  simplex,  separate  the  filaments  at  their  base  from 
the  ovary  with  a  needle,  and  examine  them  in  a  3% 
solution  of  sugar  directly  on  the  object  slide  or  in  a 
hanging  drop  in  a  moist  chamber.  They  will  live  for  a 
considerable  time  under  the  cover-glass  and  may  be  exam- 
ined with  the  highest  powers.  In  the  hanging  drop  they 
may  be  kept  for  half  a  day  in  a  living  and  developing 
state.  The  drop  should  be  made  Üat  and  shallow  in  order 
not  to  have  the  hair  settle  so  far  down  as  to  be  out  of 
reach  of  the  higher  lenses.  The  resting  nucleus  appears 
finely  punctate,  Fig.  Ill,  7,  the  under  cell;  but  if  we 
examine  it  with  a  higher  magnification,  or  if  the  cell  has 
sufiered  somewhat  from  the  influence  of  the  surrounding 
fluid  we  shall  see  that  the  nucleus  is  not  made  of  isolated 
but  of  connected  granules  bound  close  together  by  fine 
filaments,  so  that  the  whole  nucleus  represents  a  network 
enclosed  by  a  delicate  wall.  Between  these  filaments  are 
to  be  distinguished  several  nucleus  bodies  of  difl'erent  sizes. 
(350) 


NUCLEUS   DIVISION   IN    TRADESCANTIA. 


351 


The  nucleus  is  surrounded  by  a  little  protoplasm  which  is 
connected  by  threads  of  the  same  to  the  protopl  ismic  wall- 
laj'er.  This  plasma  contains,  besides  the  scarcely  distin- 
guishable microsomes,  larger  more  refractive  grains  which 
are  leucoplasts.  When  the  nucleus  is  preparing  for  self- 
division  it  increases  considerably  in  size    and  gradually 


Fig.  111.  Tradescantia  virginica.  Process  of  division  of  tlie  cells  of  the  hairs 
on  the  stamens.  1,  A  resting  nucleus  cell  below,  and  in  the  upper  cell  a  nucleus 
in  the  act  of  dividing;  2.  nucleus  showing  granular  diagonal  striae  ;5-i2,  succes- 
sive stages  of  the  process  in  the  same  cell:  3,  10  o'clock,  10  minutes;  4,  lO.iO; 
5,  10.25;  6,  10.30;  7,  10.35;  8,  10.40;  9,  10.50;  10,  11.10;  11,  11.30.  X  510. 

forms  large  grained  threads  out  of  its  finely  filamentous 
network.  The  nucleus  now  begins  to  eloufjate  and  to 
arrange  its  threads  in  a  diagonal  direction  and  nearly  par- 
allel to  each  other,  Fig.  Ill,  2.  Finally,  the  plasma  col- 
lects in  the  two  poles  of  the  nucleus.     One  may  observe 


352  DIVISION   OF   NUCLEUS   IN    TRADESCANTIA. 

all  of  these  changes  in  the  same  cell  but  it  takes  a  con- 
siderahle  time.  The  granules  become  indistinct  in  the 
filaments,  and  gradually  assume  a  homogeneons  aspect. 
The  filaments  wind  in  a  definite  but  not  always  in  a  trace- 
able way.  By  different  observations  we  may  conclude  that 
the  twist  next  makes  a  fold  at  the  equatorial  plane  of  the 
nucleus  and  the  filaments  place  themselves  parallel  to  the 
longer  axis  of  the  nuclens.  Then  the  filaments  separate  at 
the  points  where  they  bend  both  at  the  poles  and  the  equa- 
tor and  the  figure  of  the  nucleus  c(uisists  of  separate 
fragments  of  the  filaments  which  are  turned  down,  hook- 
shaped  at  the  equator.  The  next  move  is  obscure,  but  the 
stage  of  development  which  shows  itself  next  is  very  dis- 
tinctly marked.  The  fragments  of  the  filaments  are  ar- 
ranged as  shown  at  3,  in  two  separate  bundles,  straight, 
of  nearly  the  same  length,  the  ends  of  the  segments  touch- 
ing each  other  at  the  equator.  If  these  daughter  segments 
are  especially  long  they  will  be  hooked  at  their  polar 
ends.  They  are  the  same  number  in  each  bundle.  The 
change  from  the  condition  represented  in  2  has  occupied 
about  one  hour.  The  segments  appear  homogeneous, 
but  a  high  power  shows  the  surface  to  be  somewhat  beaded, 
which  betrays  a  structure  consisting  of  successively  formed 
orbicular  pieces.  At  this  moment  the  separation  of  the 
two  halves  of  the  nucleus  may  be  expected  to  take  place 
and  it  will  be  accomplished  so  rapidly  that  it  may  be 
seen  direct.  The  two  halves  of  the  nucleus  draw  apart 
longitudinally,  4.  In  five  minutes  they  are  withdrawn 
far  from  each  other,  5.  The  daughter  segments  do  not 
all  always  participate  in  this  movement  at  once,  but  often 
some  follow  the  others.  They  also  bend  up  at  the  poles 
somewhat  thicker  and  become  correspondingly  shorter,  5. 
Between  the  two  halves  then  will  appear  a  bright  trans- 
parent mass  which  will  be  increased  by  the  addition  of 


DIVISION  OF   NUCLEUS  IN  TRADESCANTIA.  353 

the  plasma  masses  which  have  heretofore  been  collected 
about  the  poles,  5  and  6. 

In  this  clear  central  mass  is  seen  a  fine  structure  which 
afterwards  may  be  seen  to  be  differentiated  into  filaments. 
About  twenty-five  or  thirty  minutes  after  the  beginning  of 
the  separation,  a  series  of  dark  points  appear  in  the  equa- 
torial plane  of  the  central  mass.  In  the  next  moment 
these  points  coalesce  and  form  a  sharply  drawn  dark  line, 
the  new  division  line.  It  consequently  arises  from  mi- 
nute granules.  They  are  microsomes  and  form  what  we 
shall  call  a  cell-plate.  It  is  equidistant  from  the  two 
halves  of  the  nuclens  in  the  middle  of  the  clear  protoplas- 
mic substance,  and  from  it  is  developed  the  new  division 
wall.  If  the  central  plasma  mass  is  large  enough  to  fill  the 
whole  diameter  of  the  cell,  we  shall  see  the  division  wall 
immediately  connected  all  around  with  the  lateral  Avail 
of  the  mother-cell.  But  if  it  only  partly  fills  the  cell  it  will 
touch  one  side  of  the  mother-cell  wall  and  the  division 
wall  will  begin  to  form  at  the  point  of  contact.  A  move- 
ment will  be  set  up  within  the  cell  which  will  brino-  the 
plasma  mass  gradually  in  contact  with  the  mother-cell 
wall  all  around,  so  as  finally  to  complete  the  division  wall. 
The  plasma  mass  draws  away  a  little  from  the  already 
formed  portion  of  the  division  wall  and  by  an  adjust- 
ment of  itself  completes  what  is  lacking  in  that  wall, 
7-9.  Dining  this  process  the  daughter  segments  bend 
their  equatorial  ends  inward  towards  the  interior  of  the 
nucleus,  7,  8.  The  ends  finally  come  in  contact  and  co- 
alesce. They  immediately  begin  to  assume  a  finely  gran- 
ular appearance,  and  with  a  high  magnification  they  seem 
to  be  a  very  thin  filament  bent  in  a  zigzag  form,  9,  and 
the  upper  cell  in  1.  The  twist  of  these  filaments  is  grad- 
ually elongated,  numerous  loops  are  formed,  and  they 
finally  anastomose  with  each  other  and  come  to  the  condi- 

23 


354  DIVISION    OF   NUCLEUS    IN   POLLEN   CELLS. 

tion  which  marks  the  end  of  our  observation,  10  and  11. 
At  the  same  time  the  daughter-cell  iucreases  in  size  and 
it  is  not  improbable  that  it  is  nourished  at  the  expense  of 
the  surrounding  cytoplasm.  The  newly-formed  division 
wall  is  slowly  nourished  by  that.  About  one  and  a  half 
hours  elapse  between  the  beginning  of  the  separation  and 
the  completion  of  the  daughter  nucleus,  and  by  this  time 
■nucleoli  are  visilile  in  the  nucleus,  11. 

Treating  with  reagents  gives  in  general  with  this  object 
not  very  satisfactory  results.  It  is  best  fixed  with  1% 
acetic  acid  in  order  to  stain  with  acetate  of  methyl  green. 
By  this  means  we  are  able  to  show  that  the  central  clear 
mass  of  protoplasm  consists  of  filaments  which  connect 
the  two  daughter  nuclei.  We  designate  them  connecting 
filaments.  The  central  ones  are  straight,  those  towards 
the  circumference  of  the  complex  being  more  and  more 
bent.  The  granules  which  form  the  cell  plate  are  found 
to  be  equatorial  enlargements  of  the  single  connecting  fil- 
aments. 

In  order  quickly  to  fix  any  of  the  various  stages  in  the 
process  of  cell  or  nucleus  division  we  will  take  the  pollen 
mother-cell  in  a  monocotyledon.  The  Liliacecß,  as  Frilil- 
laria,  Lilhim,  Alstroemeria  which  have  particularly  large 
pollen  mother-cells  and  nucleus,  are  recommended.  For 
Fritillaria  2^ersica  which  we  shall  use  here,  almost  any  spe- 
cies of  the  lily  or  amaryllis  may  be  substituted.  It  Avill 
be  found  especially  advantageous  to  select  a  species  which 
unites  in  a  single  plant  every  degree  of  development  in 
the  floral  buds,  so  that  we  may  have  exactly  that  condition 
which  we  need  for  our  examination.  We  will  select  a 
young  bud  of  the  Fritillaria  persica,  and  taking  an  anther 
lay  it  in  a  drop  of  acetate  of  methyl-green  or  gentian- 
violet.  Put  on  a  cover-glass  and  then  press  upon  it  with 
some  flat  object  till  the  compartments  of  the  anther  are 


DIVISION   OF    CELLS    IN   FRITILLAEIA   POLLEN.        355 

pressed  flat  and  emptied  of  their  contents.  The  acetic 
acid  will  flx  the  cell  contents  and  the  stain  will  color  them 
and  we  can  then  determine  whether  we  have  a  resting  nu- 
cleus or  one  in  the  process  of  division.     If  the    pollen 


Fig.  112.  FritUlarinpersica.  Division  of  the  pollen  mother-cell.  «,  ballshapecl 
foim  of  the  nucleus;  b,  the  segments  un-lei'Koing  longitudinal  division ;  c,  the  nu- 
cleus spimlle  in  pvolile;  d,  the  same  seen  from  the  poles;  e,  parting  of  the  nucleus 
plate;/,  separating  of  the  daugliter  segments;  f/,  formation  of  the  daugliter  balls 
and  tlie  cell-plate;  h,  course  of  the  filaments  in  the  daughter  nuclei;  i,  their  longi- 
tudinal elongation  and  the  formation  of  loops;  7^\  nucleus  spindle  seen  at  the  right 
in  profile  and  at  the  left  from  the  pole;  I,  separation  of  the  daughter  segments  seen 
at  the  left  from  the  pole,  at  the  right  ia  profile;  m,  granddaughter  balls,  formation 
of  the  cell-plates.  X  800. 

mother-cells  are  already  divided  into  four  daughter-cells, 
or  the  young  pollen  grains  are  already  separated  from  each 
other,  we  must  seek  for  a  younger  bud.     We  may  rec- 


356      NUCLEUS   DIVISION   IN    POLLEN   MOTHER-CELLS. 

ognize  the  young  pollen  grain,  or  the  young  pollen  mother- 
cell  by  the  thickness  of  the  colorless  cell  membrane  of  the 
latter.  We  search  till  we  tind  a  thin-walled  compound 
mother-cell  whose  nucleus  is  a  ball  of  fine  filaments,  and 
to  the  wall  of  which  are  attached  flat  nucleoli.  The  re- 
ao-ent  contracts  the  filamentous  ball  and  separates  it  from 
the  uncolored  nucleus  wall,  Fig.  112,  «,  and  one  can  see 
that  this  wall  of  the  nucleus  is  a  membranous  layer  of  the 
cell  plasma  (cytoplasm) .  We  shall  call  those  supplemen- 
tary nuclei,  secondary  nucleoli  (paranucleoli)  because  it 
occupies  a  peripheral  position  and  also  behaves  somewhat 
diflTerently  from  the  common  nucleolus.  This  paranu- 
cleolus  is  characteristic  of  all  pollen  and  spore  mother- 
cells. 

As  Ave  have  in  the  fibrillar  walls  and  the  paranueleolus 
a  preparatory  step  to  the  division  of  the  nucleus,  so  we  may 
pass  step  by  step  to  the  older  flowers.  For  fixing  we  may 
use  acetic  acid  with  methyl-green,  or  acetic  or  formic  acid 
with  gentian-violet,  or  also  picro-nigrosiu.  Preparations 
stained  with  the  last  or  with  gentian-violet  may  be  pre- 
served in  glycerine  without  further  treatment. 

A  subsequent  characteristic  stage  is  seen  in  Fig.  112  ö, 
where  segments  of  the  fibrillte  to  the  number  of  twelve  lie 
quite  evenly  distributed  about  the  wall  of  the  nuclear  cav- 
ity. They  are  stained  with  acetate  of  methyl-green,  the 
cell  cavity  remaining  colorless.  The  latter  is  filled,  in 
case  we  have  a  young  specimen,  with  homogeneous  nucleus 
sap.  An  older  stage  will  show,  in  the  nuclear  cavity,  a 
greater  or  less  number  of  delicate  cytoplasm  fibres.  The 
paranueleolus  is  but  slightly  colored  and  hangs  anywhere, 
to  a  wall  of  the  cavity  or  to  a  segment.  These  segments 
arise  from  the  fibrillse  which  we  saw  form  before  the  ball. 
The  filaments  are  shortened,  thickened,  flattened  and  finally 
are  separated  into  these  segments.     In  favorable  cases  we 


DIVISION    OF   POLLEN    MOTHER-CELLS.  357 

may  see  each  of  these  segments  split  in  two  lengthwise 
into  daughter  segments,  Fig.  112,  6,  which  form  a  Y- or  an 
X-shaped  figure. 

The  next  succeeding  characteristic  stage  is  the  formation 
of  a  nucleus  spindle,  Fig.  112,  c.  This  shows  segments 
equatorially  placed  and  deeply  stained  which  form  the 
nucleus  plate,  and  fine  unstained  spindle  fibres  which  con- 
verge towards  the  two  poles  of  the  nucleus  spindle.  The 
fibres  join  the  segments  of  the  nucleus  plate.  These  seg- 
ments are  shaped  like  a  Y  with  their  two  limbs,  following 
the  fibres,  extending  towards  the  poles.  The  nucleus  plate 
appears  from  the  poles  like  Fig.  112,  d.  The  number  of 
the  segments  in  the  plate  in  this  species  is  mostly  twelve. 
They  correspond  to  the  previously  observed  pairs  of  seg- 
ments lying  on  the  wall  of  the  nucleus.  The  nucleus  wall 
has  been  dissolved,  the  surrounding  cytoplasm  has  per- 
meated the  nuclear  cavity  and  produced  the  spindle  fibres. 
Each  segment  of  the  nucleus  plate  is  consequently  a  pair 
of  daughter  segments  the  foot  of  the  Y  being  formed  by 
the  coalescence  of  the  two  adjacent  ends  of  the  segments 
caused  by  the  action  of  the  reagent,  while  the  limbs  of 
the  letter  are  formed  by  the  two  separated  parts  of  the 
daughter  segments.  This  com[)letes  the  preparatory  stages 
of  the  division  of  the  nucleus. 

Now  the  separation  and  arrangement  of  the  segments, 
the  intermediate  stages  in  the  division  of  the  nucleus  be- 
gin. This  process  consists  of  the  separation  of  the  two 
sister  segments  of  each  pair,  while  at  the  same  time  they 
turn  their  curvature  towards  the  poles.  Fig.  112,  e.  This 
stage  is  seldom  seen  in  the  preparation  since  this  part  of 
the  process  is  quickly  run  through.  But  perhaps  the  fur- 
ther movements  of  the  dissolution  of  the  sister  segments 
belong  to  the  retrogressive  phases  of  the  parting,  the  ana- 
phases.    "We  see  such  a  condition  in  Fig.  112,  f.     The 


358  DIVISION    OF   POLLEN   3I0THER-CELLS. 

dangliter-segments  follow  the  spindle  fibres  back  quite  to 
their  polar  terminations,  where  their  ends  commingle  and 
form  the  daughter  fibrillar  balls,  Fig.  112,  g.  All  these 
conditions  we  often  find  together  in  the  contents  of  one 
anther. 

While  the  daughter  segments  collect  at  the  poles  the 
spindle  fibres  remain  intact  as  connecting  threads  between 
them,  Fig.  112,  f,  g.  Their  number  increases  by  the 
intercalation  of  new  ones  till  finally  they  form  a  barrel- 
shaped  body.  They  are  soon  distinguishable  only  at  the 
equatorial  plane,  where  by  thickening  they  form  a  series 
of  granules  which a-epresent  the  cell-plate.  Fig.  112,  g. 
The  cell-plate  extends  across  the  whole  diameter  of  the 
cell,  its  elements  commingle  and  form  a  division  wall 
which  divides  the  mother-cell  into  two  daughter-cells.  In 
the  daughter  nuclens  there  is  formed  a  bail  of  delicate 
fibres  which  run  parallel  to  the  original  direction  of  the 
daughter  segments. 

Further  preparations  show  us  that  the  fibrillte  in  the 
nuclei  of  the  daughter-cells  again  become  thicker,  Fig. 
112,  h.  They  elongate  their  twist  and  deviate  from  the  ex- 
ample in  the  first  nucleus  by  gradually  placing  themselves 
at  rischt  ansles  to  their  orioinal  direction  and  forming 
loops  at  the  equator,  Fig.  112,  i.  The  segments  are  inter- 
rupted at  the  poles  and  equator,  shorten  and  withdraw 
to  the  equator.  Thus  the  nucleus  plate  is  produced.  The 
spindle  fibres  are  recognized  w^ith  difficulty.  Fig.  112,  k, 
at  the  right.  The  segments  of  the  nucleus  plate  are 
arranged  in  the  form  of  a  wreath,  Fig.  112,  k,  left.  The 
two  nuclei  divide  in  the  same,  or  in  two  planes  at  I'ight  an- 
gfles  to  each  other.  Fiff.  112,  k,  shows  both  views.  The 
segments  of  the  nucleus  plate  divide  lengthwise  but  this 
is  not  seen  in  a  preparation  fixed  in  this  Ava3^  But  the 
daughter  segments  move  asunder  and  b}^  their  less  thick- 


.  FIXING   POLLEN-CELL   DIVISION.  359 

ness  testif}^  to  their  having  been  split,  Fig.  112,  /.  Tlie 
further  process  corresponds  to  that  of  the  mother-cell. 
The  two  cells  are  thus  divided  into  four  granddaughter 
cells  which  lie  in  the  same  plane  or  at  right  angles  to  each 
other  according  to  the  direction  in  which  the  nucleus  divis- 
ion has  taken  [)lace. 

For  a  thorough  study  of  nucleus-  and  cell-division  here 
represented,  this  method  of  fixing  is  not  satisfactory.  It 
is  better  to  put  the  material  in  absolute  alcohol.  Speci- 
mens fixed  with  chromic  or  picric  acid  or  chromic  acid 
mixture  are  not  so  good  as  those  fixed  with  alcohol.  jNIa- 
terial  laid  in  absolute  alcohol,  at  least  three  days,  may  be 
cut  lengthwise  through  the  anther  and  the  latter  put  in  a 
solution  of  safranin  in  absolute  alcohol,  after  it  has  been 
diluted  about  half  with  distilled  water  (2).  A  drop  of 
the  solution  may  be  used  on  the  slide  in  which  to  examine 
the  section  to  find  at  what  stage  of  development  the  pollen 
and  the  nucleus  division  has  arrived.  The  section  should 
lie  in  the  safranin  from  twelve  to  twenty-four  lidnrs  and 
then  taken  out  and  washed  in  absolute  alcohol  so  Ions:  as 
any  visible  color  is  given  out.  Then  lay  the  section  in  oil 
of  cloves,  or  better  in  origanum  oil,  and  as  soon  as  it  is 
saturated  with  it  transfer  to  a  solution  of  dammar  in  tur- 
pentine(dammar  dissolved  in  warm  turpentine  evaporated 
to  the  thickness  of  syrup)  when  it  will  be  preserved  un- 
changed. The  nucleus  alone  is  colored,  the  spindle  fibres 
are  but  feebly  marked.  Gentian  violet  will  give  almost 
more  beautiful  results  with  a  like  treatment  (3). 

In  order  to  make  the  spindle  fibres  visible,  we  will  lay 
a  number  of  sections  of  the  alcohol  material  in  a  very  di- 
lute solution  of  haematoxylin  —  a  few  drops  of  the  haema- 
toxylin  in  a  watch  glass  full  of  distilled  water.  The  section 
should  be  passed  from  the  alcohol  through  distilled  water 


360  STAINING  POLLEN   MOTHER-CELLS. 

into  the  staining  fluid.  This  will  avoid  the  j)resence  of  a 
precipitate.  Let  the  section  lie  several  hours  in  the  stain- 
ing fluid,  examining  it  occasionally  with  the  microscope 
to  test  the. color.  When  it  is  right,  mount  the  section  in 
glycerine.  If  the  section  has  been  excessively  colored  it 
may  be  bleached  before  putting  it  in  the  glycerine,  by 
leaving  it  for  a  considerable  time  in  water  or  by  treat- 
ment with  a  solution  of  alum.  The  over-colored  section 
may  be  toned  down  by  treatment  with  70  9^  alcohol  con- 
taining :i^  muriatic  acid,  and  then  washed  in  70%  alco- 
hol or  water,  which  contains  a  trace  of  ammonia.  But 
this  method  requires  especial  care.  Far  more  beautiful 
haematoxylin  preparations  which  are  equal  to  the  safranin 
stains  are  obtained  by  passing  the  section  stained  with 
haematoxylin  through  absolute  alcohol  into  oil  of  cloves  or 
oil  of  lavender  and  from  this  into  Canada  balsam  dissolved 
in  chloroform.  The  section  will  need  to  remain  but  a  short 
time  in  the  alcohol  and  in  the  volatile  oil. 

One  may  quickly  obtain  an  instructive  preparation  from 
alcohol  material  by  staining  with  diamant-fuchsin-iodide- 
green  (4).  Make  a  stain  of  diamant-fuchsin  and  iodide- 
green  in  50%  alcohol.  Pour  the  iodide-green  S(jlution  in 
a  vessel  and  slowly  add  the  diamant-fuchsin  until  the  fluid 
takes  on  a  conspicuous  violet  color.  Put  the  section  of 
the  anther  in  a  drop  of  the  solution  for  a  minnte  on  the 
slide  and  then  inclining  the  slide  let  the  fluid  run  off",  and 
absorb  it  with  blotting  paper.  Then  add  a  drop  of  gly- 
cerine, arrange  the  section  and  put  on  the  cover-glass. 
The  cytoplasm  will  be  colored  red,  the  nucleus  blue  and 
the  paranucleolus  red.  For  sharpness  of  outline  it  stands 
next  to  a  good  preparation  stained  with  safranin  and  haem- 
atoxylin. It  may  be  mounted  in  Canada  balsam  or  gum- 
mastic.     The  former  is  to  be  preferred  on  most  accounts 


DIVISION   OF   POLLEN    CELLS   IN   HELLEBORUS.      361 

but  has  the  disadvantage,  for  use  with  homogeneous  im- 
mersions that  the  oils  employed  dissolve  it.* 

If  gum  mastic  is  to  be  used  the  delicate  object  must  be 
protected  from  the  pressure  of  the  cover-glass.  This  may 
be  done  by  drawing  some  ridges  of  the  gum  on  the  slide 
where  the  edg^es  of  the  cover-sflass  would  come  and  letting 
it  partly  dry  immerse  the  object  in  the  gum  l^etween  them 
and  lay  on  the  cover-glass.  Cement  by  subsequent  ap- 
IDlication  of  the  dissolved  gum,  or  of  gold  size.  A  small 
drop  of  wax  applied  to  the  slide  Avill  serve  the  same  pur- 
pose, f  In  the  longitudinal  section  of  the  anther  the 
mother-cells  will  be  found  in  various  successive  stages  of 
development,  which  the  observer  will  find  very  useful. 

For  a  study  of  the  development  of  the  pollen  mother- 
cell  of  the  dicotyledons,  we  will  take  a  plant  from  the  lia- 
nunculacece  or  the  Papaveraceae, — in  this  case  Hellehoriis 
foetidus.  In  a  floral  I)ud  which  with  its  style  measures 
about  8  or  10  mm.  the  successive  anthers  from  within  out- 
ward will  give  all  the  stages  in  the  process  of  division. 
Treat  the  anther  in  the  same  way  and  with  the  same  fixing 
and  staining  fluids  as  we  did  the  FritiUaria  and  we  shall 
get  the  same  appearance  only  that  the  pollen  grains  are 
smaller.  After  the  first  step  in  the  division  of  the  mother- 
nucleus  a  cell  plate  will  form  among  the  connecting  fila- 
ments, but  again  dissolve  while  the  nucleus  prepares  for 
the  second  step.  This  fully  agrees  with  the  first  in  dis- 
tinguishing the  process  from  that  of  the  FritiUaria.  The 
nuclear  pairs  are  united  by  connecting  filaments.  The 
four  nuclei  arrange  themselves  in  the  spherical  mother- 

*This  difficulty  may  be  obviated,  howevev.  by  running  a  ring  of  sliellac  or  other 
cement  not  soluble  in  the  homogeneous  fluid  around  the  edge  of  the  cover-glass. 
—A.  B.  H. 

fTlius  abbreviated  the  author.  But  very  shallow  shellac  ring-cells  put  on  with 
a  turn-table  and  a  hair  pencil  will  serve  all  purposes  much  better.— A.  B.  H. 


362    DEVELOPMENT  OF  POLLEN  CELLS  IN  HELLEBORUS. 

cell  in  the  four  corners  of  a  tetrahedron,  Fig.  113,  A. 
Connecting  fibres  run  freely  in  all  directions  through  the 
cytoplasm  between  the  four  nuclei.  Thus  four  new  bun- 
dles of  fibres  are  added  to  the  two  already  existing  in  each 
of  which  is  produced  a  cell-plate.  The  latter  are  distinct, 
but  the  connecting  filaments  are  seen  onW  under  the  most 
favorable  conditions.  The  cell-plates  have  a  circular  quad- 
rilateral form.  They  meet  within  the  mother-cell.  On 
the  thick  wall  of  the  mother-cell  are  six  inner,  somewhat 
projecting  ridges,  A,  to  which  the  cell  plates  attach  them- 
selves with  their  outer  edges.  Cellulose  Avails  are  soon 
formed  of  the  plates  and  the  mother-cell  is  divided  into  four 

tetrahedrically-a  r  r  a  n  ije  d 
^  daughter-cells.  These  four 

cells  soon  have  walls  of 
their  own,  while  the 
mother-cell  wall  is  dis- 
solved. 

Fig.  113.    ffeUeborus   fmtidus.    Pollen  ThoSC     plants     ill    wllicll 

mother-cell  at  y4  in  the  act  of  dividing:  at      ^^^\   ,i*    ••,.,,     „^.,0,      ^.,,.K„  ,«- 

„^  „    ,.  .,  ,  ..,,„  °'        cell-divisiou    was    earliest 

i)  fully  divided.  X  510. 

observed  belonged  to  the 
species  Cladopliora  glomerala,  whose  structure  we  have 
already  studied  and  know  to  be  muUinuclear  (5).  Cell- 
division  is  not  necessarily  preceded  by  a  division  of  the 
nucleus.  Each  daughter-cell  is  provided  with  a  number 
of  nuclei  which  may  further  increase,  so  that  the  division 
of  the  cell  and  of  the  nucleus  may  go  on  independently  of 
each  other.  Cell-division  may  be  found  going  on  at  any 
hour  of  the  day,  but  again  we  may  often  seek  for  it  in 
vain.  If  one  is  found,  others  are  likely  to  be  in  the  same 
culture.  One  may  easil}'  recognize  the  process  of  division 
since  the  place  where  the  division  Avail  is  to  come  is  marked 
by  a  bright  ring.  The  process  (6)  begins  Avith  a  feeble 
annular  collection  of  cytoplasm  near  the  middle  of  the 


DIRECT   AND    INDIRECT    CELL-DIVISION.  363 

length  of  the  cell.  The  chlorophyll  layer  dnnvs  back 
correspondingly.  The  beguinuig  of  the  divit^ion  wall 
sliows  forth  now  as  a  sharp  line.  It  projects  ledge-like 
into  the  cell-space  and  presses  the  chlorophyll  layer  inward. 
The  inconspicuous  ring  of  cytoplasm  remains  on  the  inner 
edge  of  the  projecting  ledge.  On  both  sides  of  the  form- 
ino-  division  wall  cell-sap  collects  between  the  chlorophyll 
]axev  which  is  being  pressed  inward  and  the  delicate  mem- 
branous hn^er ;  thence  the  colorless  ring  in  the  dividing 
cell.  The  chlorophyll  contents  are  finalh*  bisected  and  the 
diaphragm-like  wall  fully  closes  up  in  the  middle  and 
makes  a  complete  division  wall.  The  chlorophyll  contents 
remain  for  some  time  at  some  distance  from  the  newly 
formed  wall  but  gradually  draw  near.  The  nuclei  are  too 
small  to  allow  the  details  of  their  division  to  be  satisfac- 
torily seen.  The  various  steps  in  the  division  may  be 
easily  fixed  with  1%  chromic  acid,  but  they  are  seldom 
met  with. 

All  those  processes  of  division  of  the  nucleus  which  are 
connected  with  the  formation  of  filaments  are  classed  to- 
gether as  indirect  nuclear  division,  and  stand  in  opposition 
to  the  direct,  which  consists  of  a  simple  bisecting  of  the 
nucleus.  The  latter  process  is  often  seen  in  the  older 
cells  of  the  more  highly  organized  plants, — as  in  the  un- 
usual case  of  the  rapidly  growing  internodal  cells  of  the 
Chanicece  (7). 

For  the  observation  of  the  direct  nuclear  division  of  the 
older  cells,  the  older  hxternodes  of  Tradescantiavh'ginica 
furnish  a  very  favorable  object.  A  longitudinal  section 
examined  in  water  Avill  usually  show  a  large  number  of 
them,  Fig.  114,  A.  The  nucleus  exhibits  its  original  con- 
tents but  more  or  less  irregularly^  contracted  into  several 
segments  of  difterent  form  and  size.  In  many  cases  the 
pieces  have  been  fully  separated  and  lie  together  or  at  a 


364 


DIRECT   NUCLEUS   DIVISION. 


little  distance  from  each  other.  There  may  be  eight  or 
ten  of  them  of  various  sizes,  and  these  again  may  increase 
by  self-division.  While  the  nucleus  may  be  found  in  the 
act  of  division  in  almost  all  the  elements  of  the  section, 
it  ma}^  be  best  observed  in  those  of  the  medullary  paren- 
chyma. The  thin-walled  elements  of  the  vascular  bundles 
"which  have  a  nucleus  show,  besides,  streaming  protoplasm 
most  beautifully.     The  nuclei  may  be  quickly  fixed  with 


0^ 


,.^ 


ßm 


:'S-\ 


Ica: 


Fig.  114.  Tradescantia  virginica.  Nuclei  of  the  older  intei-iiodes  in  the  act  of 
direct  jjarting.  A,  iu  living  state ;  B,  after  treatment  with  acetate  of  methyl-green. 
X540. 

acetate  of  methyl-green  when  they  come  out  very  distinctly, 
Fig.  ]  14,  B. 

Finally,  taking  our  highest  lenses  to  aid  us,  we  will 
enter  upon  a  question  the  solution  of  which  is  of  the 
greatest  moment  for  the  complete  understanding  of  plant 
bodies.  It  treats  of  the  mutual  connection  of  the  proto- 
plasmic cell  bodies  of  the  plant,  which  form  a  single  con- 
tinuous whole  (8).    Select  Rhamnus  frangula  and  having 


PLASMA  CONNECTIOXS  OF  CELLS.        365 

removed  the  periderm  from  a  stem  about  a  centimeter 
thick  make  a  delicate  tanofential  section  throuo-h  the  sfreeu 
rind.  Put  the  section  in  water  and  direct  attention  to  the 
chlorophyll-containing  bast-parenchyma,  which  consists  of 
rectangular  cells  tangentially  extended.  The  walls  are 
consideral)ly  thickened  and  provided  with  both  large  and 
small  unbordered  pits,  the  latter  so  narrow  as  to  be 
scarcely  distinguishable  (9).  Besides  the  bast-paren- 
chyma the  spindle-shaped  groups  of  cells  of  the  medullary 
rays  are  seen. 

Xow  make  a  new  section  in  the  same  direction,  put  it  on 
a  cover-glass  and  add  a  drop  of  concentrated  sulphuric 
acid.  After  a  few  seconds  immerse  the  whole  in  a  glass 
full  of  water  and  wash  the  section  thoroughly  and  quickly. 
Stain  with  an  aqueous  soluti(m  of  aniline  blue,  wash  with 
water  and  examine  in  dilute  glycerine.  Picric-aniline  blue 
may  advantageously  be  used  instead  of  the  other.  Pre- 
pare it  by  dissolving  to  saturation  picric  acid  in  5^^  alcohol 
and  add  aniline  blue  till  the  solution  takes  on  a  blue-areen 
color. 

Use  the  highest  powers,  a  homogeneous  immersion  if 
possible.  The  effect  of  the  acid  is  satisfactory  if  the  walls 
of  the  bast-parenchyma  are  so  much  swollen  that  they  show 
about  the  same  diameter  as  the  contracted  cell  body.  The 
middle  lamella  is  also  swollen  which  makes  the  object 
still  more  favorable  for  our  investigation.  The  contracted 
plasma  body  is  beautifully  stained  with  the  aniline  blue. 
The  outlines  of  the  single  plasma  bodies  of  the  rind-paren- 
chyma cells  are  smooth  on  those  surfaces  which  border 
upon  the  cell  walls  which  are  provided  with  very  fine  pores. 
But  when  they  adjoin  walls  with  wider  pits  they  are  pro- 
vided with  processes  more  or  less  thick.  These  processes 
correspond  in  neighboring  cells.  If  noAV  we  carefully  ex- 
amine the  inclosing  membrane  between  two  particularly 


366  PLASMA   CELL   CONNECTIONS. 

broad  oppositely-placed  processes,  ayg  shall  find  a  num- 
ber of  exceedingly  fine,  granular  filaments  stretched  be- 
tween them.  They  are  the  plasma  threads  by  which  living 
plasma  bodies  communicate  with  each  other.  The  outer 
filaments  of  such  a  complex  are  bent  and  remind  one  strik- 
ingly of  the  connecting  fihuuents  which  join  two  sister 
nuclei.  Where  the  adjacent  walls  of  two  cells  are  smooth, 
we  find  that  the  middle  layers  of  the  cell  wall  are  perme- 
ated throughout  their  whole  extent  by  filaments  which  by 
a  very  considerable  swelling  of  the  cell  wall  will  be  sep- 
arated from  both  the  plasma  bodies,  but  in  case  of  less 
swelling  will  still  be  connected  with  them.  These  fila- 
ments are  somewhat  swollen  and  spindle  shaped  in  the 
middle.  In  especially  favorable  cases  the  spindles  will 
appear  to  be  interrupted  in  the  middle  and  the  two  halves 
connected  by  an  extremely  delicate  granular  thread.  This 
appearance  is  seldom  seen.  Generally  the  plasma  bodies 
do  not  all  show  us  their  common  plasma  connections,  but 
rather  those  which  have  in  no  way  been  injured  in  cut- 
ting the  section,  and  which  have  been  suddenly  fixed  by 
the  acid.  The  injured,  or  those  not  fixed  with  sufficient 
rapidity,  have  withdrawn  their  plasma  filaments. 

Those  cell  walls  which  are  penetrated  with  fine  filaments, 
have,  with  the  filaments,  an  appearance  which  suggests  the 
case  of  the  nucleus- and  cell-division  where  the  connectins: 
filaments  served  as  starting  points  for  the  division  wall, 
and  which,  now  remaining  intact,  maintain  a  communica- 
tion between  the  two  cells  (10).  By  the  formation  of 
broader  pitted  surfaces,  the  connection  is  afterwards  main- 
tained only  within  them,  but  that  a  direct  connection  exists 
between  neighboring  cells  b}'  means  of  plasma  processes, 
seems  to  be  now  pretty  well  demonstrated. 

It  is  much  easier  to  demonstrate  this  proposition  by  some 
recentl}"  discovered  facts  involving  the  presence  of  proto- 


INTERCELLULAR   PLASMA.  367 

plasmic  masses  in  the  intercellular  spaces  (11).  For  this 
investigation  take  a  year  old  branch  of  Ligustrum  vulgare 
(12).  Put  it  in  alcohol  for  some  dajs  in  order  to  harden 
the  cell  body.  Since  by  cutting  a  fresh  object  the  cell 
contents  might  escape  into  the  intercellular  spaces,  and 
vitiate  the  result,  make  a  delicate  section  which  shall 
include  the  primary  rind  and  lay  it  in  potassic  iodide  of 
iodine  solution.  The  rind  is  formed  of  rounded,  pretty- 
Avell-thickened  cells  with  intercellular  spaces  of  various 
sizes  between.  These  are  either  filled  or  covered  with  a 
substance  which  takes  the  same  yellow-brown  color  as  the 
protoplasmic  cell  contents.  The  effect  may  be  heightened 
if,  after  the  removal  of  the  iodine  solution,  one  v/ill  add  a 
little  dihite  sulphuric  acid  (acid  2  parts,  water  1),  which 
will  produce  a  slight  swelling,  and  the  characteristic  blue 
color  of  the  walls.  The  yellow-brown  color  of  the  proto- 
plasm in  the  cell  and  in  the  intercellular  spaces  will  stand 
out  very  clearly.  Still  more  instructive  will  be  a  lonoi- 
tudinal  section,  the  cells  being  longitudinally  elongated 
and  some  of  the  intercellular  spaces  of  considerable 
length. 

Notes. 

(1)  See  for  this  lesson,  Strasburger,  Zellb.  u  Zellth.,  in  Aufl. ;  Flera- 
ming,  Zellsubst.  Kern,  u.  Zellth.,  Strasburger;  die  Controversen  der 
Kerntheilung.     lu  the  latter  work  the  literature. 

(2)  Flenimiug,  Arch.  f.  Micros.  Auat.,  Bd.  xix,  p.  317. 

(3)  Flemmiiig,  Zellsub.  Kern,  etc.,  p.  .S84. 

(4)  For  double  staining  of  tissue  these  coloring  substances  were  first 
proposed  by  J.  Macfarlane,  Trans.  Bot.  Soc.  Edinb.  Vol.  xiv,  p.  190. 

(5)  Von.  V.  Mohl.  im  Jahre  1835  Dissert.  Abgedr.  in  Flora  1837,  ff. 

(6)  Strasburgr,  work  before  quoted,  p.  203. 

(7)  Johow,  Bot.  Ztg.,  1881,  sp.  728,  Strasburger;  Ueberden  Tlieil- 
ungsvorg.  d.  Zellk.,  p.  98,  Auch.  Arch.  f.  mikr.  Anat.  Bd.  xxi.  Liter- 
ature there. 


368  LITERATURE    OF   LESSON. 

(8)  For  the  general  view,  see  Strasburger,  Bau  u.  Wachsthum  der 
Zellhaute,  p.  246,  1882. 

(0)  This  object  was  recommeuded  by  Russow,  the  method  of  inves- 
tigation by  Gardiner,  Phil.  Trans.  Roy.  Soc,  Part  iii,  1883,  p.  821,  ff. 

(10)  See  Strasburger,  worlv  last  quoted  p.  248,  and  Russow  Stzber. 
d.  Dorpaternaturf.  Gesell.  1882. 

(11)  See  Russow,  1.  c,  p.  19,  Berthold  Ber.  d.  deut.  botan.  Gesell. 
II,  Jahrg.  p.  20. 

(12)  Recommended  bj' Berthold,  1.  c. 


INDEX. 


[  Abbi-eviations :  d  =  development,  r  =  reaction,  s  =  structure,  u  =  use-] 


Abbe  illarainating  apparatus,  2. 

"  "  "    u.,     218. 

Acei',  yellow  autumn  leaves,  43. 
Acetic  acid,  u.,  27,  28,  46,  76,  162, 
168. 
"         "       1%  354. 
"       2%  327. 
"     38'/„  48. 
Aconitum  napellus,  s.  of  seed  bud, 

322. 
Acorus  calamus,  s.  of  root,  131. 
Adjustment,  coarse,  10;   fine,  10. 
Adonis  flammeus,  color  bodies  of 

blossom,  42. 
uEcidium  berberidis,  s.  of  hyme- 
nium,  253. 
"  "  spermagonia,  255. 

uEsculus    hippocastanum ,   glandu- 
lar tuft,  80. 
Agar-agar,  u.,  234. 
Agaricus  campeslris,  191. 
Air,  removing  from  object,  41,  45, 

69,  328,  337. 
Air-bubbles,  recognizing,  11. 
Albuminous  bodies,  r.,  21. 

"  crystals  of    Berthol- 

letia  excelsa,  28 ;  Bicimis  com- 
munis, 26. 
Alcanna  tincture,  u.,  27,  112. 
Alcoholabsolute,u.,27,  35,  39,  45, 
55,78,  89,  98,  99,  113,  149, 
225,226,  227,293,299,324, 
334,  337,  359,  367. 
"     40%,  225. 
"     50%,  112,  360. 
"     70%,  360. 
24 


Aleuron  grains  in  Bertholletia  ex- 
celsa, 28 ;  Lupinus  alba,  25 ; 
Pisum  sativum,  19 ;  Bicinus 
communis,  26;  r.,  20. 

Alis7na  plantago,  s.  of  the  fruit, 
336;  of  the  germ,  337;  of  the 
seed,  338. 

Allium  cepa,  s.  of  root,  129. 

Aloe  nigricans,  stomata,  65. 

Althcea  rosea,  pollen  grains,  313. 

Alum  in  aqueous  solution,  u., 
196. 

Alum-carmine,  88. 

Ammonia,  u.,  196,  360. 

Ampelopsis  hederacea,  red  autumn 
leaves,  43. 

Anabcena  azolloe,  208. 

Anaptychia  ciliaris  =  Physcia  cil- 
iaris,  apothecium,  260;  sper- 
magonium,  261;  thallus,  192. 

Aneimia  fraxinifolia,  s.  of  epi- 
dermis, 68. 

Aniline,  sulphate  of,  58. 

"        blue,   u.,    114,   123,  126, 
226,  365. 
green,  0,001%  u.,  227. 

Annual  rings  in  trees,  102. 

Anther,  s.  and  d.  of  in  Hemero- 
callisfulva,  306;  Lilium,  309; 
Tradescantia  virginica,  310. 

Antheridium  of  Marchantia  poly- 
morpha,  264 ;  Mnium  hornum, 
269;  Peranosporacece,  250; 
Polypodium  vulgare,  282  ;  Pol- 
ytriclnim  juniperinum,  271; 
Vaucheria  sessilis,  244. 
(369) 


370 


INDEX. 


Antirrhinum    majus,   cell-sap    of 

corolla,  41. 
Apical  cell  of  Equisetum  arvense, 

167f ;    Metzgeria,   189 ;    Pteris 

critica,  179. 
Archegonium  of  Marchantia  poly- 

morpha,  266;  Mnium  hornum, 

271 ;     Picea      vulgaris,     301 ; 

Polyjyodium  vulgare,  284. 
Aristolochia  sipho,  s.  of  stem,  98. 
Arrow  root.  East  ludia,  13. 
"  "     West  India,  14. 

Aspidium  filix-mas,  sporangia,280. 
Autumnal  colors,  brown,  43;  red, 

43;  yellow,  43. 
Avena  sativa,  starch  grains,  15. 
Bacteria,  preparing  the  material, 
214. 

"       cilia,  215. 

"      culture,  228. 

*'       permanent  mounting  of, 
220. 

"      developmental  forms  of, 
223. 

"      mould  membrane,  215. 

"      germination  of,  232. 

"       nomenclature,  223. 

"      in  pock-lymph,  221. 

"      spore  building,  216,  231. 

"      methods  of  staining,  214, 
220,  225. 

"       of  tnberculosis,  224. 

"       investigation  of,  in    the 
tissue,  227. 

"      cell  contents,  221. 

"       Zoogloea,  214. 
Bacterium  subtile,  229. 
Bacillus    tnberculosis,  permanent 
preparation,  225. 

"         staining,  225. 
Beggiatoa  alba,  222. 
Bean  meal,  13. 
Bertholletia    excelsa,   albuminous 

crystals,  28. 
/ 


Beta  vulgaris,  cell  structure  of,  45 ; 

sugar  test  in,  48. 
Bismai'ck  bi«own,  u.,  266. 
Blood-serum,  u.,  234. 
Borax    carmine,  20,    89;    Grena- 

cher's,  196;  Thiersch's,   196. 
Butomus  rindulatus,  ovary,  318. 
Calluna  vulgaris,  pollen,  315. 
Cambiform  cells,  96. 
Cambium,  interfascicular,  100. 
Camphor,  u.,  301. 
Canada  balsam,  u.,  225,  360. 
"  "in  chloroform,  89. 

"      r     "     in  turpentine,  89, 

220,  226. 
«'  "in  xylol,  228. 

Cane  sugar,  as  irritating  medium 

for  spermatozoid  of  mosses, 

286. 
Capsella  bursa  pastoris,  s.  and  d.  of 

germ   and    seeds,  332;  s.    of 

seed-coat,  333. 
Carbolic  acid,  u.,  302,  313,   314, 

337. 
Carmine,  Beale's,  196. 

"      and  acetic  acid,  98. 

"      ammoniacal,Hoyer's,  196. 
Cedar  oil,  220. 
Cell,  multiuuclear,  37. 
Cell-division  in    Cladophora,  S62 ; 

in  anther  of  Fritillaria,  354 ; 

in  Hellebore,  361 ;  in  Trades- 

cantia,  351;  by  periclinal  and 

anticlinal  walls,  164;  at  acute 

angles,  181. 
Cell-sap,  blue,    41 ;     yellow,   41 ; 

purple,  41;   rose-colored,  42, 

43. 
Cellulose,  r.  on,  46,  52. 
Cell  walls,   s.   in  endosperm    of 

date,  54;  in  seed  of  Ornitho- 

galum,    53 ;     in     Pinnularia, 

202 ;  in  Pinus  sylvestris,   57 ; 

middle  lamella  of,  53 ;  lamina- 


INDEX. 


371 


tion  of,  53 ;    striation  of,  50, 
52;    lignified,  r.  of,  58,    113; 
suberized,  s.,  148;  r.,  148f. 
Cementiug  the   preparation,  361. 
Ceric  acid  reaction,  148. 
Cheiranthus  alpiuus,  hairs,  72. 

"  cheiri,  hairs,  71. 

Chelidonium  majus,  vascular  bun- 
dles, 97. 
"  "  milk  tubes,  97. 

Cherry-wood  extract,  u.,  58. 
Chloral  hydrate,  u.,  39,  313,  314. 
Chloroform,  u.,  27. 
Chlorophyll  grains,  s.  in  prothal- 
liura  of  fern,  39 ;  in  Funaria 
hygrometrica,  38. 
Chlorophyll  dividing,  38. 
Chloriodide  of  zinc,  u.,  46,  52,  53, 
58,  63,  67,  82,  116,  121,    148, 
193,  229,  254. 
Chromic-acetic  acid,  1  %,  196. 
Chromic  acid,  u.,  58,  148,  206. 
"      "    5%,  227. 
"      "    1  %,  196,  363. 
"      "    20  %,  205. 
"      "    25%,   306,    313, 
315. 
Citron  oil,  u.,  313,  314. 
Citrus  vulgaris,  adventive  germs 
of,  348. 
"  "    s.  of  fruit,  344f. 

"  "     d.  of  fruit,  346. 

Cloves,  oil,  u.,  225,  227,  228,  313, 

359. 
Cladophora    glomerata,   194,  237, 
362. 
"  "  pyrenoids,  195; 

swarm-spores,  237 ;    nucleus, 
195;  cell-division,  362. 
Collenchyma,  98. 
Collodion,  324. 

Color  bodies  of  flower  of  Adonis 
flammeus,  42 ;  Tro- 
pueoluiii  majus,  40. 


Color  bodies  of  root  of  Daucus 
carota,  42. 

Column  of  microscope,  6. 

Conducting  cells,  84,  87,  119. 

Coneof  gymnosperms,s.  and  mor- 
phological meaning  of,  297. 

Copper  acetate,  u.,  48. 
"      sulphate,  u.,  47. 

Coralline  (in  30  %  carbonate  of 
soda  solution),  u.,  85,  90,  93, 
95,  97,  113,  121,  131,  137. 

Cork,  u.,  in  making  sections,  62. 
"  s.  and  d.  in  Cytisus  labur- 
num, 148;  Quercus  suber,  149; 
Hibes nibrum,  lid;  Sambucus 
nigra,  147;  r.  of,  148;  stain- 
ing, 148 ;  s.  of  cell  wall,  148. 
"  u.  in  cutting  sections,  332, 
337. 

Cover-glasses,  4. 

Cucurbito  pepo,  vascular  bundles, 
123 ;  plasma  streaming  in 
hairs,  35;  pollen  grains,  314. 

Culture  methods  for  bacteria,  214, 
228. 
"       apparatus,  232. 
"       by  division,  233;  in  gela- 
tine, 233 ;  by  dilution,  233. 

Cuprammonia,  u.,  52,  219. 

Curcuma  leucorhiza,  starch,  13. 

Cuticle,  r.,  63. 

Cutin,  r.,  58. 

Cystids,  258. 

Cytisus  laburnum,  s.  and  d.  of 
cork,  148. 

Dahlia  variabilis,  s.  of  bulb,  49. 

Dammar  gum,  u.,  220,  225,  359. 

Delphinum  consolida,  coloring 
matter  of  petals,  42 ;  ajacis, 
ovary,  317. 

Diamant-fuchsiu,  iodine-green, 
360. 

Diaphragms,  cylindrical,  6. 

Diphenylamin,  u.,  49. 


372 


INDEX. 


Draccena  rubra,  s.  of  stem,  92. 

Drawing  prism,  u.,  2,  31 ;  Abbe's, 
3,  31 ;  with  two  prisms,  3,  32; 
board,  3 ;  microscopic  objects, 
12,  31. 

Drosera  rotundifolia,  digestive 
glands,  79. 

Dust,  removing  from  the  prepara- 
tion, 23. 

East  India  arrow  root,  13. 

Echeveria,  wax  coating,  80. 

Eleagans  aiiyxistifolia,  scale  hairs, 
75. 

Elder- pith,  7;  u.,  GO,  152,  156,  183, 
193,  248,  278,  332. 

Embryo,  s.  and  d.,  Alisma  plan- 
tago,   337;    Caj)sella    bnrsa 
2)astoris,  332;   Ficea  vulga- 
ris, 302. 
"  adventive  in  orange,  348. 

Embrjo  sac,  s.  and  d.  in  Capsella 

bursa  pastoris,  S36  ;  Monotro- 

pa    hypopitys,    324;    orchids, 

327;     Torenia   asiatica,    330. 

"  egg-apparatus,  326. 

Endochrome  plates  of  Pinnularia 
viridis,  204. 

Endoderm,  s.  in  root  of  Acorus 
calamus,  130;  Allium  cepa, 
130 ;  Irisflorentina,  132 ;  outer, 
131. 

Endosperm,  d.  in  Monotropa  hypo- 
pitys, 327. 

Epidermis,  s.  in  Aloe  nigricans, 
66;  Iris  floreiitina,  60f. 

Epidermoidal  layerj  131. 

EpijMctis  palustns,  ovary,  320. 

Equisetum  arvense,  s.  of  stem,  168. 
"  "    vascular  bundles, 

169. 
"  "     apical  cell,  167. 

Ether,  u.,  27,  149. 

Eucalyptus  globosus,  wax  coating 
of,  81. 


Euphorbia  helioscopia,  starch,  15. 
"  splendens,  starch  grain, 
15. 

Evonymus  japonicus,  d.  of  shoot, 
from  vegetative  cone,  164. 

Fagus  silvatica,  s.  of  leaf,  155. 

Fehling's  solution,  u.  and  prepara- 
tion, 47. 

Ferric  alum,  u.,  360. 
"     chloride,  51. 
"      sulphate,  u.,  51. 

Fertilization  process  in  Marchan- 
tia  polymoipha,  267 ;  Mono- 
tropa hypopitys,  326;  Feran- 
ospora,  250;  Ficea  vulgaris, 
299  ;  Folypodium  vulgare,  285 ; 
Vaucheria  sessilis,  244. 

Fibro-vascular  cord,  84. 

Fibrils  of  Physcia  ciliaris,  193. 

Filiform  apparatus,  329. 

Finding  again  a  definite  place  in 
the  preparation,  231. 

Fixing  cell  contents  with  chromic 
acid,  196 ;  chromic-acetic  acid, 
196 ;  picric  acid,  196. 

Foot  of  the  microscope,  6. 

Fritillaria  persica,  cell  and  nucle- 
us, division  in,  354. 

Fruit,  s.  in  Alisma  plantago,  337 ; 
Citrus  vulgaris,  344;  F)'unns 
domestica,  341 ;  Fyrus  malus, 
342  ;  d.  in  Citrus  vulgaris,  346. 

Fuchsin,  u.,  215,  225. 

Funaria  hygrometrica,  chlorophyll 
grains  in,  38. 

Gall-apple,  s.,  51 ;  tannin  contents 
of,  51. 

Gelatine,  u.,  233,  315. 

Gentian  violet,  u.,  39,41,215,  227, 
359. 
"  "     and     formic    acid, 

356. 
"  "in     aniline     water, 

227. 


INDEX. 


373 


Gentian  violet  with  acetic  acid, 
354,  35G. 

Ginkgo  biloba,  autumnal  yellow 
color  of,  43. 

Glandular  tufts  of  ^sculns  hippo- 
castanum,  80;  of  Bumex  pa- 
tentia,  78. 

Glass  bells,  high  and  low,  5. 
"    disks,  5. 
"    rods,  5. 
"    tubes,  5. 

Globoids,  in  aleuron  grains  of  Ber- 
tfiolletia  excelsa,  28 ;  Bici7ius, 
26. 

Gloeocapsa  polydermata,  s.  of  cell, 
211. 

Gloxinia  hyhrida,  embryosac,  328. 

Glycerine,  u.,  18,  32,  55,  89,  99,  108, 

114,  123,    12G,  167,  197, 

281,       332,     360,     365. 

"      jelly,    u.,    89,     197,     324. 

"      gum,  u.,  183. 

Glucose,  48. 

Gold  size,  u.,  361. 

Gonidia  of  Physcia  ciliaris,    193. 

Gum,  u.,  284,  332. 

Haematein-ammonia,stainingwith, 
196,  208 ;  preparation  of,  197. 

Haematoxylin,  u.,  28,  324,  359. 

"  Bohmer's,    u.,  196; 

Grenacher's,  u.,  196. 

Hairs,  s.,  in  Cheiranthus  alpimis, 
72 ;  Ch.  cheiri,  71 ;  Matthiola 
annua,  72 ;  Verhascum  nigrum, 
73 ;  V.  thapsiforme,  74  ;  Viola 
tricolor,  72 ;  bristle  of  Urtica 
dioica,  77;  stinging  of  the 
same,  76;  glandular,  of  Dro- 
sera rotundifolia,  79;  oi  Pri- 
mula sinensis,  77;  human,  u., 
23;  horse,  u.,  241;  scale,  of 
Eleagnns  angttstifolia,  75 ;  of 
Shepherdia  Canadensis,  74. 

Hand-vice,  5;  u.,  18,  53. 


Helleborus  fcetidus,  cell    and  nu- 
cleus division  in,  362. 
Hemerocallis   fulva,   s.  and  d.  of 
anther,  306;  ovary,  319;  pol- 
len, 304. 
Hippuris  vulgaris,  vegetative  cone, 

161. 
Hordium  vulgare,  vegetative  cone 

of  root,  174. 
Horsehair,  u.,  241. 
Hyacinth,  ovary,  319. 
Hyaloplasm,  30. 

Hydrocharis    morsus    ranoe,  root- 
hairs,  35. 
Hydroids,  57. 

Hypochlorine-reactiou,  195. 
Hypoderm,  85. 

Illuminatiug  apparatus,  Abbe's,  2. 
"      u., 
218. 
Intercellular  passages,  82 ;  plasma 

contents  of  same,  367. 
Inulin,  testing,  50 ;  spherical  crys- 
tals of,  50. 
Iodine  in  alcohol,  u.,  16,  39. 
"      "  glycerine,  u.,  26. 
"      "  potassic  iodide  solution, 
u.,   16,  26,  46,  195,  240, 
244,  259,  261,   265,  284, 
311,  367. 
"      "  water,  16,  41. 
"     green,  u.,  88,  311. 
Iris  florentina,  s.  of  leaf,  88. 
"  "     endoderm     of     root, 

132. 
"  "     epidermis      of     leaf, 

60. 
"  "    vascular     bundle    of 

leaf,  88. 
"    germanica,     leucoplasts    and 
starch  in  rhizome,  44. 
Lathyrus,    formation     of    pollen 

tube,  315. 
Lavender  oil,  u.,  360. 


374 


INDEX. 


Leaf,  s.  in  Fagtis  silvatica,  155 ; 
Muiuni  undulatum,  183 ;  Buta 
graveolens,  152 ;  Sphagnum 
acutifolium,  184. 
"  arrangement  and  function  of 
chloropliyll- containing  cells, 
158. 
•'  influence     of   liglit    on    tlie 

structure  of,  157. 
"    kinds  of  tissue  in  assimila- 
tion and  ti'anspiration  tissue, 
159;  nerve  parenchyma,  159. 
"    mechanical    adjustment    of, 
157. 
Lenticels  of  Samhucus  nigra,  146. 
Leptothrix  buccalis,  223. 
Leucoplasts,    in    Iris    germanica, 
44 ;  in  staminate  hairs  of  Tra- 
descantia,    30;    in   Verbascum 
nigrum,  41 ;   Tradescantia  vir- 
ginica,  65. 
Ligustrum  vulgare,  protoplasm  in 

the  intercellular  spaces,  367. 
Lily,  s.    of  ovary,  319;  d.  of  an- 
ther, 309. 
Lime  oxalate,  in  cells  of  Beta  vul- 
garis, io;  Iris  florentina,  91; 
Bosa  semperflorens,  76 ;  reac- 
tion of,   45;    phospliate,    u., 
199;  sulphate,  u.,  199. 
Linden  wood,  u.,  332. 
Litpinus  albus,  aleuron  grains,  26. 
Lycopodinm    complanatum,    s.    of 

stem,  142. 
Maceration  mixture,  Schulz',  u., 

106,  148. 
Magnesia,  sulphate  of,  u.,  199. 
Magnifying  glass,  2;  aplanatic,  3, 
Malic  acid  as  an   irritant  of  the 

spermatozoa  of  ferns,  285. 
Malva  crispa,  pollen,  314. 
Maranta  arundinacea,  starch,  14. 
Marchantia    polymo7pha,     s.     of 
thallus,  185f ;  s.  of  reproduc- 


tive organs,  263f ;  process  of 
fertilization,  267f;  gemmae, 
263;  oil  bodies,  187;  rhizoids, 
188;  sporogouium,  268. 

Mastic  gum,  u.,  361. 

MaWiiola  annua,  hairs,  72. 

Mechanical  system,  85. 

Medullary,  crown,  103;  rays,  in 
Binus  sylvestris,  113,  115; 
rays,  secondary,  102. 

Methyl-blue,  u.,  220. 

Methyl-green,  u.,  46,  and  formic 
acid,  356;  acetate  of,  u.,  20, 
46,  54,  312,  354,  356. 

Methyl-violet,  u.,  39,  41,  225,  226  ; 
BBBBB,  u.,  225. 

Metzgeria  furcata,  s.  of  thallus, 
188f. 

Mica,  u.  205. 

3Iicrococcus  vaccinae,  221. 

Microsomes,  30. 

Microtome,  u.,  62,  171. 

Milk-tubes,  s.  in  Chelidonium  ma- 
jus,  97 ;  sap,  97. 

Millon's  reagent,  u.,  20. 

Mnium  /iorttJtm,  an  the  ridia,    269; 

archegonia,  271 ;  blossom, 

269;  sporogonium,  272. 

"     undulatum,   s.  of  leaf,   183; 

of   stem,  181 ;    movement  of 

water  in  central  cord,  182. 

Monotropa  hypopitys,  d.  of  embry- 
osac,  325. 

Moist  chamber,  7. 
"  "        for  hanging  drop, 

230,  236. 

31orchella  esculenta,  hymeniura, 
259 ;  epiplasm,  259. 

Mounting  medium,  u.,  89,  197. 

Mucilage,  from  cellulose,  and 
from  starch,  94 ;  staining,  94. 

Mncor  Mucedo,  sporangia,  246 ; 
zygospores,  247. 

Muriatic  acid,  u.,  58,  76,  197. 


INDEX. 


375 


Muriatic  acid,  10%,  u.,  225. 
"     30%,  u.,  197. 
"  "     i%  in  70%  alcohol, 

u.,  360. 

Needles,  4;  holders,  4. 

Nerium  oleander,  s.  of  epidermis, 

68. 
Nigrosin,  u.,  79,94;    picrate  of, 
230. 

Nitrate,  raicrochemical  reaction 
of,  49. 

Nitrite  microchemical  reaction 
of,  49. 

Nostoc  ciniflonum,  209. 

Nucleus,  division  of,  in  Fritillana 
persica,  355;  Hellehorus  foz- 
tidus,  362 ;  Tradescantia  Vir- 
ginica,  351fl",  363 ;  permanent 
preparation  of,  360;  direct, 
363 ;  fixing  and  staining  at 
various  stages  and  with  differ- 
ent reagents,  356,  359,  360; 
indirect,  363. 

Nucleus,  of  Penicillium  crnsta- 
ceum,  252;  Saccheromyces  cere- 
visice,  208;  Spirogyra,  200;  in 
hairs  Tradescantia  virginica, 
350;  in  the  pollen  of  the 
same,  311 ;  staining,  20;  behav- 
ior of,  in  fertilization,  327. 

Nutation  of  Oscillaria,  211. 

Nutritive  fluid  for  fresh  water 
alga;,  199. 

Objective  for  homogeneous  immer- 
sion, 1,  2;  u.  of,  218. 
"    for  water    immersion     1; 

u.  of,  218. 
"     micrometer,  3. 

Object  slide,  4;  form  of,  4. 

Ocular,  erecting,  2. 

Oil,  essential,  i*.,  27;  fatty,  r.,  27; 
drops,  26;  olive,  u.,  28;  ori- 
ganum, u,,  359. 

Onothera  biennis,  pollen,  312. 


Oogonium,  of  Peranosporoe,  250; 
Vaucheria  sessilis,  243. 

Orchids,  embryosac  and  fertiliza- 
tion, 327. 

Origanum  oil,  u.,  359. 

Ornithogalum  umbellatum,  s.  of 
cell  wall  of  seed,  53. 

Oscillaria,  movement  in,  211 ;  hab- 
itat, 210;  cell  structure,  210. 

Ovary,  s.  in  Bntomus  umbellatus, 
318  ;  Delphinium  ajacis,  317 ; 
Epipactis  palustris,  320;  Hem- 
erocallis,  319;  hyacinth,  319; 
lily,  319  ;  Primula,  320 ;  tulip, 
319;  monocarpous,  317;  poly- 
carpous,  317;  inferior,  320; 
superior,  317. 

Over-colored  preparations,  treat- 
ment of,  196. 

Ovule,  anatrophic,  323 ;  campy- 
lotropic,  336 ;  chalaza,  323 ; 
d.  and  s.  of,  in  Aconitum  na- 
pellus,  322;  Capsella  bursa 
pastoris,  335 ;  Citrus,  348 ; 
Picea  vulgaris,  299 ;  funicu- 
lus of,  322;  micropyle,  323; 
nucellus,  323 ;  raphe,  322  ;  sec- 
tion of,  323. 

Pceonia,  formation  of  pollen 
tubes.  315. 

Palisade  cells,  152. 

Papaver  rhoeas,  s.  of  petal,  160. 

Para  nucleolus,  356. 

Pencil,  hair,  5. 

Penicillium  crustaceum,  asci,  251 ; 
mycelium,  250;  habitat,  250; 
nucleus,  252. 

Permanent  preparations,  making, 
21,  87. 

PeranosporecR,  antheridium,  250 ; 
fertilization,  250;  oogonium, 
250. 

Perosmic  acid,  u.,  27,  28,  227, 
264. 


376 


INDEX. 


Petals,  s.  in  Papaver  rhoeas,  160; 
in  Verbascum  nigrum,  160. 

Phaseolus  vulgaris,  starch,  13. 

Phelloderm  in  Bibes  rubrum,  150. 

Phellogen,  146. 

Pliloroglucin,  58. 

Phoenix  dactylifera,  s.  of  endo- 
sperm cell  walls,  54. 

Physcia  ciliaris,  apothecium,  260; 
spermagonium,  261 ;  thallus, 
192. 

Phytophthora  infestans,  conidia, 
247. 

Picea  vulgaris,  archegonium,  301 ; 
fertilization,  299 ;  embryo  sac, 
300;  seeds,  302;  female 
bloom,  299f. 

Picric  acid,  u.,  196,  208. 

Picro-alcohol,  u,,220;  picro-aui- 
line-bliie,  u.,  88,  365;  -nigro- 
sin,  u.,  88,  356;  -sulphate,  u., 
230;  carmine,  u.,  227. 

Finnularia  viridis,  movement  of, 
205  ;  endochrome  plates,  204  ; 
preparation  of  skeleton,  205 ; 
dividing,  204;  cell  membrane, 
203. 

Piniis  sylvestris,  s.  of  stem,  108f ; 
s.  of  male  flowers,  289;  pol- 
len, 291 ;  bordered  pits  in 
wood,  54;  female  flowers, 
296. 

Pisum  sativum,  s.  of  stem,  18. 

Pits,  bordered,  in  Pinus  sylvestris, 
54;  simple  in  Agaricus  cam- 
pestris,  192;  in  Beta  vulgaris, 
45;  one-sided,  104,  109;  clos- 
ing membrane  of,  56;  torus 
of,  56. 

Placenta,  free  central,  of  Primu- 
lacece,  320. 

Plasmolysis  in  staminate  hairs  of 
Tradescantia,  34. 

Pleurosigma  angulatum,  206. 


Pollen  grains,  s.  in  Acacia,  315; 
Althea  rosea,  313 ;  Azalia,  315 ; 
Calluna  vulgaris,  315 ;  Cur- 
cubita,  314 ;  Erica,  315 ;  Hem- 
er ocallisfnlva,  304;  Leucojum, 
312;  Malva  crispa,  314;  Mi- 
mosa, 315;  (Enothera  bien- 
nis, 312  ;  Pinus  sylvestris,  291 ; 
Rhododendrons,  315. 

Pollen  grains  ;n  Taxus  baccata,29S 
Tradescantia  virginica,  310 
making  transparent,  313 
tubes,  315;  nucleus  of,  311. 

Polypodium  vulgare,  antheridia, 
270. 

Poplar  wood,  u.,  332. 

Potassium,  chlorate  of,  u.,  106. 
"  bichromate,  u.,  52. 

"  acetate,  u.,  162,  168. 

"  nitrate,  u.,  199. 

"  solution  of,  u.,  16,  39, 

76,  97,  132,  148,  162,  166,  227, 
253,  272,  288,  295,  313,  329, 
334,  337. 

Preparations,  preservation  of 
stained,  197;  removal  of  air 
from,  23;  made  under  the 
microscope,  23f. 

Primula,  ovary,  320. 

"  sinensis,  glandular  hairs, 77. 

Prism,  erecting,  2. 

Procambium,  165. 

Prothalliura  of  Polypodium  vul- 
gare, 281. 

Protococcus  viridis,  206. 

Protonema,  182. 

Protophloem,  84. 

Protoplasm,  circulation  of,  36;  in- 
diflerence  layer,  36,  37 ;  inter- 
cellular spaces,  367 ;  rotation 
of,  36;  connection  of  in 
neighboring  cells,  364; 
streaming  in  leaf  of  Vallis- 
neria  spiralis,  36 ;  in  hairs  of 


INDEX. 


377 


young  sprouts  of  pumpkin, 
35;  in  root  of  Hydrocharis 
morsus  ranee,  35 ;  inNitella,  37. 

Protoxylera,  84. 

Prunus  domestica,  s.  of  fruit,  341. 

Pteris  aquilina,  s.  of  rhizome, 
13Sf ;  Pteris  critica,  d.  of  root, 
179. 

Puccinia  graminis,  255. 

Pyrenoids  of  Cladophora  gloine- 
rata,  195. 

Pyrus  comrmmis,  s.  of  cell  in  fruit, 
46. 
"       malus,  s.  of  fruit,  342. 

Quercus  suber,  s.  of  cork,  149. 

Banunculusrepens,  s.  of  adventive 
root,  133 ;  of  vascular  bun- 
dle, 95,  96. 

Raphides,  93. 

Razor,  4,  18. 

Resin,  r.,  112. 

Resin  ducts,  s.  in  Pinus  sylvestris, 
112,  115. 

Ehammis  frangula,  plasma  con- 
nections between  neighbor- 
ing cells,  364. 

Bihes  rubrum,  phellodenn,  150. 

Ricinus,  aleuron  grains,  26. 

Roots,  s.  of,  iu  Acorus  calamus, 
131 ;  in  Allium  cepa,  129  ;  Ba- 
nuuculus  repens,  133 ;  Taxus 
baccata,  134. 

Root-cap  of  gyiTiuospernis,  175; 
of  Ilordeum  vulgare,  174. 

Bosa  semperfloreus,  s.  of  spine, 
75. 

Roseaniline,  sulphate,  u.,  225. 
"      violet,  Hanstein's,  u.,79. 

Bumex  patientia,  glandular  tufts 
of,  78. 

Biissula  rubra,  257. 

Buta  graveolens,  s.  of  leaf,  151f. 

Saccharomyces    cerevisice,    sprout- 
ing, 207;  nucleus,  208. 
25 


Saccharum  officinarum,  wax  coat- 
ing of,  81. 

Safranin,  u.,  87,  168,  228. 
"        in  alcohol,  225. 

Salt,  common  cooking,  199. 

Sambuctis  nigra,  cork  and  phello- 
derm,  145f. 

Schulze's  maceration  mixture,  u., 
106,  148. 

Sclerenchyma,  47. 

Scolopendrium  vulgare,  sori,  278 ; 
sporangia,  280. 

Scalpel,  4. 

Secondary  nuclei,  356. 

Section  making,  18,  54 ;  with  very 
thin  objects.  183. 

Seeds,  s.  in  Alisma  plantag o,  337; 
Capsella  bursa  pastoris,  332 ; 
Picea  vulgaris,  302;  Prunus 
domestica,  342;  Tnticum  du- 
rum, 21 ;  methods  of  investi- 
gating, 332. 

Selaginella  Martensii,  sporangia, 
287;  spores,  288;  vegetative 
organs,  287. 

Seed  coat,  s.  in  Capsella  bursa 
pastoris,  333. 

Serum  of  cattle  and  sheep's  blood, 
u.,  234. 

Sheath  of  vascular  bundle,  84. 

Shepherdia  Canadensis,  scale 
hairs,  74. 

Shoemakers'  globe,  u.,  219. 

Sieve-tubes  of  cuctirbitapepo,  124 ; 
Lycopodium  complanatum, 
144;  Pinus  sylvestris,  114; 
Tilia  parvifolia,  119;  Zea 
Mays,  84.  Callus  plates  in, 
87,  114,  115,  126,  127;  stain- 
ing the  same,  87,  114;  con- 
tents of  tubes,  87. 

Silicious  skeletons  of  diatoms, 
preparation  of,  205. 

Simplex,  description  of,  23 ;  u.,  23. 


378 


INDEX. 


Soda,  solution  of,  48. 

Sodic  sulphate,  u.,  222. 

Solanum  tuberosum,  starch  in 
tuber,  8,  10. 

Sperm  nucleus,  327. 

SperniMgoniuni  of  JEcidium  ber- 
beridis,  255 ;  of  Physcia  cilia- 
ns,  261. 

Spermatia  of  ^cidium  berberidis, 
255  ;  of  Physcia  ciliaris,  261. 

Sperniatozoids  oi Marchantia poly- 
inorpha,  264  ;  Milium  hormtm, 
270 ;  Polypodium  vidgare,  283 ; 
Vaucheria,  244;  fixing,  in 
ferns,  284. 

Spine  of  rose,  3,  75f. 

Spirochaeta  plicatilis,  222. 

Spiroytjra,  copulation  of,  236f. 
"     majuscula,  culture  of,  199. 
'<  "  cell    structure, 

199. 

Sphagnum  acutifolium,  s.,  184. 

Sponge  parenchyma,  152. 

Sporangia,  s.  in  Asjndium  filix- 
mas,  280f;  3Iucor  mucedo, 
246;  Scohipiendriun  vulgare, 
279 ;  Selaginella  Ilartensii, 
288. 

Spores  of  ^cidium  berberidis,  254 ; 
Physcia  ciliaris,  261 ;  Bacteria, 
216,  231 ;  Marchantia  poly- 
marpha,  269 ;  Mnium  hornum, 
274;  Morchella  esculenta,  259; 
Mucor  mucedo,  246 ;  Scolopen- 
drium  vulgare,  280;  Selagi- 
nella 3Iartensii,  288. 

Spores,  basidia,  of  Eussula 
rubra,  258 ;  macrospores,  288  ; 
microspores,  288 ;  swarm 
spores  of  Cladophora  glomera- 
ta,  237,  240 ;  Vaucheria  sessilis, 
240,  241 ;  teleutospores  of 
Puccinia  graminis,  256 ;  ure- 
dosporcs  of  the  same,  255. 


Sporidia  orFucciiiia  graminis,  257. 

Sporogonium,  s.  in  Marchantia 
pjolymorpha,  268,  in  Mnium 
hornum,  272. 

Spring  clips,  6. 

Spring  sheaih  of  microscope  body, 
6. 

Staining  Bacteria,  220f,  225f. 

"  double,  87;  the  cell  con- 
tents with  various  media, 
196. 

Staphylea,  formation  of  the  pollen 
tube,  315. 

Starch  grains,  s.  in  various  plants, 
8,  13,  14,  15,  17,  44;  testing 
in  mixtures,  38;  lamination 
of,  9  ;  relation  to  heat,  17  ;  to 
reagents,  17;  compound  and 
semi-compound,  12. 

Steel  forceps,  4. 

Stem,  s.  in  Aristolochia  sipho,  98f ; 
Lycopoditimcomplanatum,  142 ; 
Pinus  sylvestris,  108;  Tiliapar- 
vifolia,  118f. 

Stereids,  85. 

Stomata,  s.  in  AloS  nigricans,  65f ; 
Aneimia  Jraxinifolia,  68 ;  Iris 
ßorentina,  60 ;  Tradescantia 
virginica,  64;  T.  zebrina,  65; 
mechanism  for  movement  in, 
62,  63;  guard  cells,  62,  63,  64. 

Stone  cells  of  the  pear,  47. 

Style,  319. 

Suberine  reactions,  148f. 

Sugar,  testing,  in  the  pear,  47: 
in  the  sugar  beet,  48f;  solu- 
tion, u.,  84,  315;  2,%,  u.,  325, 
328,  329;  r.,  Barfoed's,  48; 
Fehling's,  47. 

Sulphur  in  bacteria  cells,  222. 
"        carbonate,  u.,  222. 

Sulphuric  acid,  45,  46,  53,  57,  63, 
67,  130,  205,  315,  365. 

Sunflower-pith,  u.,  61. 


INDEX. 


379 


Tannic  acid  in  gall  apple,  51. 

Tartrate  of  potash  and  soda,  u. 
47. 

Taxus  baccata,  s.  of  root,  134f; 
arillus  of,  296;  blossoms  of, 
male,  292,  female,  293  ;  pollen, 
293. 

Test  objects,  205. 

Thallus  of  Physcia  ciliaris,  192; 
Marchantia  polymorpha,   185. 

Thickening  growth,  secondary  in 
Aristolochia  sipho,  98  ;  in  roof 
of  Taxus  baccata,  134  ;  abnor- 
mal in  Draccßiia  rubra,  92. 

Thuia  occidentalis,  vegetative 
cone  of  root,  175f. 

Tilia  parvifoUa,  s.  of  stem,  118f. 

Torenia  asiatica,  fertilizing,  329. 

Iradescantia  virgitiica,  plasma 
streaming  in  staminate  hairs 
of,  31f;  pollen,  310,  315;  sto- 
mata,  64;  cell  and  nucleus,  di- 
viding in,  351,  363;  zebrina, 
stomata,  65. 

Triticitm  durum,  starch,  14. 

"  vulgare,  s.  of  fruit  and  seed, 
21. 

Tropceolum  majus,  color  of  blos- 
som, 40;  water  pore,  69. 

Tube  of  the  microscope,  6. 

Tulip,  ovary,  319. 

Turpentine  oil,  u.,  220,  225. 

Urtica  dioica,  stinging  hairs  and 
bristles  of,  76,  77. 

Vallisneria  spiralis,  protoplasm 
streaming  in  leaf,  36. 

Vaucheria  sessilis,  process  of  fer- 
tilization, 244;  reproductive 
organs,  243;  swarm  spores, 
241 ;  nucleus,  242. 

Va.scular  bundle  cylinder,of  roots, 
129. 
"  "     course  of,  in  petals 

of     Verbascum     ni- 
grum,  160. 


Vascular  bundle,  s.  of,  in  leaf  of 
7m  florentina,  88 ;  in  petiole 
of  Polypodium  vulgare,  141 ; 
Scolopendrium  vulgare,  141; 
stem  of  Chelidonium  majus, 
97  ;  Curcubita  pex)o,  123 ;  Dra- 
coena  rtibra,  92 ;  Pteris  aquili- 
na,  189;  Ranunculus  repens, 
95  ;  Zea  Mays,  82  ;  in  the  root 
of  Acorus  calamus,  131 ;  of 
Alium  cepa,  129;  of  liannncu- 
lus  repens,  133 ;  bast  portion 
of,  84;  bi-coUateral,  123;  col- 
lateral 84;  ending  of,  160;  be- 
longing to  the  leaves,  162; 
vascular  part,  84;  closed,  82; 
wood  part,  84;  open,  95; 
phloem,  84;  protophloem,  84; 
protoxylera,  84;  sieve  part, 
84;  belonging  to  the  stem, 
162;  staining,  85,  86,  88;  xy- 
lem,  84. 

Vegetative  cone,  s.  of,  in  stem  of 
various  plants,  161-167;  s.  in 
roots  of  various  plants,  173, 
175,  177,  179;  making  trans- 
parent, 162,  166,  167 ;  struct- 
ural elements,  163;  staining, 
167;  methods  of  investigat- 
ing, 161f,  166;  cell-division  in, 
164. 

Vegetative  point  of  Metzgeria  fur- 
cata,  189. 

Verbascum  nigrum,  vascular  bun- 
dles in  petals,  160;  hairs  of 
corolla  and  stamens,  73  ;  cell- 
sap  of  petals,  41. 

Vesuvin,  u.,  220. 

Vessels  of  Cucurbita  pppo,  123. 

Vinca  major,  colored  sap  in  blos- 
som, 41  ;  sclerencliyma  hbres 
in  stem,  52. 
Viola  tricolor  grandiflora,  hairs  of 
corolla,  73. 

Watch  glasses,  5. 


380 


INDEX. 


Water  pores  of  Tropceohim  majus, 

69. 
Wax,   u.,   361,  coating  on  Eche- 

veria  globosa,  80;  Eucalyptus 

globosus,   81 ;    on   Saccharum 

officinarum,  81. 
Wheat  flower,  starch,  14. 
White  of  an  egg,  u.,  301. 
Wood,  s.  in  Aristolochia  sipho,  102 ; 

Finns    sylvestris,    108 ;     Tilia 

parvifolia,  118;  disintegration 

by  maceration,  106. 


Wood-parenchyma,  86. 

Wood,  r.,  58,  113. 

Xylol,  u.,  220,  228. 

Zinc  racli  for  slides,  4,  5. 

Zooglcea,  214. 

Zea  Mays,  s.  of  vascular  bundle, 

82  f. 
Zygospores  of  Mucor  niucedo,  247. 


APPENDIX, 


Staining  with  carmine. — Carmine  solutions  stain  diffusely,  but  one 
may  obtain  a  distinct  color  in  the  nucleus  by  laying  the  stained  prep- 
aration for  some  time  in  50%  to  70  %  alcohol  containing  0.5  to  1  %  mu- 
riatic acid,  or  in  glycerine  to  which  has  been  added  0.5  %  muriatic 
acid. 

Preparation  of  Beale's  carmine. — Pour  2.3  cc.  concentrated  ammonia 
upon  0.6  g.  pulverized  carmine.  After  dissolving  the  carmine  let  it 
stand  an  hour  and  then  pour  it  iuto  a  mixture  of  66  cc.  vpater,  47.5  cc. 
concentrated  glycerine  and  19  cc.  absolute  alcohol.  Mix  and  filter 
after  a  short  time. 

Grenadier's  alum- carmine. —'YioW  for  ten  to  twenty  minutes  a  1-5  % 
aqueous  solution  of  common  alum  with  i  to  1  %  of  pulverized  carmine 
and  filter  after  cooling.     To  this  add  a  trace  of  carbolic  acid. 

Grenarher's  borax-carmine. — Dissolve  2-3  %  carmine  in  a  4  %  aque- 
ous solution  of  borax  and  dilute  with  a  like  quantity  of  70  %  alcohol. 
Filter  after  a  considerable  time. 

Thiersche's  borux-carminc.  Dissolve  4  parts  borax  in  56  parts  dis- 
tilled water.  To  this  solution  add  1  part  carmine,  and  then  mix  1  vol- 
ume of  the  same  with  2  volumes  of  absolute  alcohol  and  filter. 

Carminate  of  ammonia — Hoyer's  neutral.  Heat  in  a  sand  bath  1  g. 
carmine  in  about  1-2  cc.  strong  ammonia  solution  and  6-8  cc.  of  wa- 
ter, till  the  excess  of  ammonia  has  evaporated  which  is  shown  by  the 
appearance  of  small  bubbles  and  the  fluid  becoming  a  bright  red.  Fil- 
ter the  precipitate  from  the  nearly  neutral  fluid  after  cooling,  and  to 
this  add  4-6  volumes  of  strong  alcohol.  This  causes  the  deposit  of  a 
bright  red  precipitate,  which  filter  out  and  preserve.  This  powder  is 
dissolved  in  water  when  needed  and  may  be  preserved  by  adding  1-2  % 
chloral  hydrate. 

Chlor-iodide  of  zinc. — Dissolve  metallic  acid  in  pure  muriatic  acid 
and  evaporate  to  the  consistency  of  sulphuric  acid  by  the  constant 
addition  of  the  zinc.  To  this  add  all  the  potassic  iodide  that  will  dis- 
solve and  as  much  metallic  iodine  as  it  will  take  up. 

Hoyer's  mounting  fluid. — For  aniline  preparations.  Fill  a  wide-necked 
glass  two-thirds  full  with  selected  white  pieces  of  gum  arable  and  fill 
up  the  vessel  with  an  ofliciual  solution  of  potassic  acetate  or  ammo- 
nia. By  frequent  shaking  the  gum  will  dissolve  in  a  few  days.  Filter 
through  blotting  paper.     For  carnune  or  haeraatoxylin  preparations  a 

(381) 


382  APPENDIX. 

several  per  cent  solution  of  chloral  hydrate  to  which  has  been  added 
5-10  %  of  glycerine  should  be  substituted  for  the  ammonia  or  the  po- 
tassic  acetate.  If  the  fluid  becomes  tuibid  after  a  while  it  sliould  be 
again  filtered. 

Glycerine-jdly. — To  one  part  of  the  finest  French  gelatine,  which 
has  been  soaked  for  two  hours  iu  six  parts  by  weight  of  water,  add 
seven  parts  of  pure  glycerine,  and  to  each  100  g.  of  the  mixture  add 
1  g.  concentrated  carbolic  acid.  Warm  and  stir  for  ten  to  fifteen  min- 
utes till  all  flocculeuce  caused  by  the  addition  of  the  acid  disappears. 
Finally  filter  while  warm,  through  finest  glass  wool  previously  washed 
and  still  damp  with  distilled  water. 

Glycerine-gum. — 10  g.  gum  Arabic,  10  g.  water,  40-50  drops  of  gly- 
cerine. 

Hcematoxylin  staining  fluid,  Boehmer's. —  Dissolve  0.35  g.  haenia- 
toxylin  in  10  g.  absolute  alcohol,  and  to  this  solution  add  drop  at  a 
time  from  a  solution  of  0.1  g.  alum  in  30  g.  distilled  water,  till  a  beau- 
tiful blue  violet  color  is  obtained. 

Grenacher's. — No.  1,  A  saturated  solution  of  hseraatoxylin  in  absolute 
alcohol.  No.  2,  A  saturated  solution  of  ammoniacal  alum.  Mix  4  cc. 
of  No.  1  in  about  150  cc.  of  No.  2.  Let  it  stand  a  week  iu  the  light. 
Filter  and  add  22  cc.  glycerine  and  25  cc.  methyl-alcohol.  It  should 
stand  some  time  before  using,  to  settle. 

Cuprammonia. — The  bright  green  precipitate,  which  cupric  hypo- 
sulphate  gives  with  dilute  solution  of  ammonia,  should  be  filtered  and 
washed,  and  while  still  moist  poured  into  concentrated  ammonia,  in 
which  it  dissolves  with  the  development  of  heat.  After  cooling, 
crystals  of  hyposulphate  of  cuprammonia  will  form.  The  fluid  con- 
tains only  cuprammonia  from  which  the  crystals  should  be  filtered 
out,  and  the  solution  kept  in  black  bottles  or  in  the  dark. 


3(o  4 


^GA 


